Showing papers in "Progress in Nucleic Acid Research and Molecular Biology in 1993"

Initiation of transcription by RNA polymerase II: a multi-step process

Abstract: Publisher Summary This chapter discusses the initiation of transcription of protein-coding genes. The RNA polymerase II(RNAPII) of the yeast Saccharomyces cerevisiae has been a useful prototype in the study of eukaryotic RNAPII, as features such as function and subunit structure have been highly conserved. The yeast RNAPII is composed of 11 polypeptides with apparent masses ranging from 220 to 10 kDa. This is in contrast to eukaryotic cells, which contain three distinct RNA polymerases, each containing from 8 to 14 polypeptides and responsible for transcribing its own set of genes: RNA polymerase I (RNAPI), which transcribes ribosomal RNA; RNAPII, the RNA polymerase of protein-coding, or class 11, genes; and RNAPIII, which transcribes 5-S rRNA and tRNA genes. In this chapter, only RNAPII is discussed, and only as it pertains to transcription initiation.

Evolution of Ca(2+)-dependent animal lectins.

Abstract: Publisher Summary This chapter discusses the evolution of Ca 2+ -dependent animal lectins Animal lectins have been identified in various contexts, both as a result of direct searches for proteins with selective sugar-binding activities, and less systematically in the course of investigation of biological recognition processes In spite of the enormous diversity of animal lectins, analysis of their primary structures reveals that most fall into relatively few categories In all cases, sugar-binding activity is associated with discrete protein modules, of 115-140 amino acids, termed carbohydrate-recognition domains (CRDs) Three major groups of animal lectins (P, S, and C-types) contain CRDs with distinct sequence motifs Animal lectins, grouped together are based on structural considerations also share certain important functional properties The mannose-6-phosphate receptors (P-type lectins) and thiol-dependent p-galactoside-binding lectins (Stype lectins) each have relatively restricted specificity for a particular type of sugar structure The C-type lectins, in contrast, are characterized not by a common type of sugar ligand, but by a shared dependence on Ca2 + for sugar-binding activity

tRNA structure and aminoacylation efficiency.

Abstract: Publisher Summary This chapter discusses the role of tRNA structure in the recognition process with synthetases and on the implications for aminoacylation efficiency. Many examples are taken from our own research on several specific aminoacylation systems, for example aspartate, histidine, valine, but concepts are presented more globally with reference to the complete set of aminoacylation systems. It emphasizes on the importance of tRNA-like structures for understanding the interaction of canonical tRNAs with synthetase. Although tRNA-like molecules found in some plant viral RNAs do not participate in protein synthesis, they represent interesting natural mutants to be compared to canonical tRNAs. This is also the case of tRNAlike structures found in some messenger RNAs as well as of bizarre tRNAs from mitochondria . In addition, competition and kinetic effects may also contribute to the overall specificity of the various aminoacylation systems; the balance between the concentration of tRNAs and synthetases would be essential for ensuring optimal specificity. According to this view, individual aminoacylation systems do not work at their optimal chemical efficiency, but work instead to assure optimal discrimination among the different aminoacylation systems. Such a balance may be perturbed under certain physiological or pathological conditions. Finally, this chapter discusses a comparison of recent results with previous observations, and show how old concepts established phenomenologically can now be tested more explicitly.

Regulation of Repair of Alkylation Damage in Mammalian Genomes

Abstract: Publisher Summary This chapter discusses the current understanding of the molecular basis of the regulation of alkylation damage repair in mammals. Such molecular studies were not possible until the recent success in the cloning of the alkylation repair genes. This chapter first explains the basic mechanisms of repair proteins. The availability of nucleic-acid and antibody probes for many of the alkylation repair genes and proteins, and elucidation of the structure and identification of the regulatory elements of these genes, provide an opportunity for a comprehensive understanding of regulation of alkylation damage repair. The prospect of large-scale production of the human alkylation repair proteins in E . coli for the subsequent determination of their structure by X-ray crystallography and NMR looks quite good. In contrast to inhibition of repair genes at the level of their expression, these genes could also be inactivated in cultured cells by homologous recombination. Starting with mutations in repair genes of pluripotent embryonic stem cells, repair-deficient or repair-negative mice could be generated. Such animals may make excellent models for mutagen, carcinogen, and aging studies.

Structure and action of mammalian ribonuclease (angiogenin) inhibitor.

Abstract: Publisher Summary This chapter discusses the recent studies of the mammalian ribonuclease inhibitor, and in particular, human placental ribonuclease inhibitor (PRI) Far less is known about this protein inhibitor of ribonucleases than about protein inhibitors of proteinases, for which a vast literature exists Nevertheless, recent studies have revealed distinctive properties of this family of proteins, properties of interest from the point of view of understanding their relation to the inhibition of the activities of the mammalian RNase superfamily of enzymes, including angiogenin in particular Angiogenin exhibits specific, saturable binding to calf pulmonary endothelial cells It stimulates phospholipases C and A, in endothelial cells at concentrations as low as 01 ng/ml, but is not an endothelial cell mitogen The 35% identity of the angiogenin primary structure to that of bovine pancreatic ribonuclease A (RNase A) is a most unexpected feature Three residues catalytically essential in RNase A (Lys-41, His-12, and His-119) are fully conserved in angiogenin Importantly, the catalytic activity of angiogen is distinct from that of RNase A or other RNases, and this in turn, distinguishes it from all other angiogenic factors These findings provided a unique opportunity to examine naturally occurring ribonuclease inhibitors for their antiangiogenic properties, and this resulted in the finding that the human protein RNase inhibitor abolishes both the angiogenic and ribonucleolytic and phospholipase C-stimulating activities of angiogenin

Global regulation of mitochondrial biogenesis in Saccharomyces cerevisiae.

Abstract: Publisher Summary This chapter describes the regulation of expression of nuclear genes that encode proteins involved in mitochondria biogenesis, particularly, the regulation exerted by carbon source and oxygen. It reviews features of mitochondrial proliferation in relation to cell division, and it discusses possible mechanisms through which the yeast cell adjusts the biosynthesis of mitochondrial components relative to metabolic requirements and cellular growth rate. In baker's yeast, S. cerevisia, synthesis of a respiration-competent mitochondrion is controlled mainly by environmental stimuli, such as the availability of oxygen and the type of carbon source. In the presence of oxygen and the absence of a fermentable carbon source, transcription of nuclear genes encoding components of the respiratory chain and proteins of the mitochondrial transcription/translation machinery is induced four to tenfold. Recent biochemical analyses reveal the presence in yeast of yet another class of regulatory proteins. In contrast to the low-abundant specific regulators like GAL4 and the HAP proteins, which exert their effects on relatively small families of genes, a small group of highly abundant sequence-specific DNA-binding proteins is involved in diverse regulatory events, such as activation and repression of transcription, initiation of DNA replication, and chromosome maintenance. Well-characterized members of this family of multifunctional regulators are ABF1, CPF1, and RAP1 are described in the chapter.

Mechanism of Action and Regulation of Protein Synthesis Initiation Factor 4E: Effects on mRNA Discrimination, Cellular Growth Rate, and Oncogenesis

Abstract: Publisher Summary This chapter discusses the progress in laboratory and those of others in understanding one point at which regulation of the rate of protein synthesis occurs, the mRNA-binding step. It focuses on the structure, function, and regulation of protein synthesis initiation factor 4E (eIF-4E). Initiation of protein synthesis is a complex and highly regulated process, and there are numerous mechanisms by which regulation occurs, both of a global nature and mechanisms targeted for individual mRNAs. This chapter explains on only one factor and one step in protein synthesis, eIF-4E and the binding of mRNA to ribosomes. Thus, one may think of eIF-4E as a proto-oncogene and consider translational routes to oncogenesis, to complement and broaden current transcriptional models. In fact, some of the poorly translated proto-oncogene mRNAs listed in table that is given in the chapter, encode transcription factors, such that a general stimulation of translation would be expected to produce a disproportionate increase in transcription factor levels.

Molecular Biology in the Eicosanoid Field

Abstract: Publisher Summary This chapter describes the molecular biology in the eicosanoid field. Molecular biology made its main entrance to the eicosanoid field, when two groups simultaneously reported the cloning of the cDNA for leukotriene A 4 hydrolase. Subsequently, the field has expanded considerably to include many interesting findings with the lipoxygenases, prostaglandin-synthesizing enzymes, and prostanoid receptors. Availability of the genes for these enzymes should allow targeting strategies to discern potential functions. Clear information on the regulation and significance of leukotriene formation by 5-lipoxygenase, 5-lipoxygenase activating protein, and leukotriene A 4 hydrolase is still lacking. The recent discovery of the second form of cyclooxygenase has reopened a wide interest in the academic and pharmaceutical communities in prostaglandin research. The differential regulation of the two cyclooxygenase forms and the development of selective isoform inhibitors will be the areas of intensive research. Cloning of the different members of the eicosanoid receptor family should facilitate our unravelling of the mechanisms involved in the action of this interesting class of arachidonate metabolites known as eicosanoids.

Mammalian 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase - a Bifunctional Enzyme That Controls Glycolysis

Abstract: Publisher Summary This chapter explains the enzyme system that catalyzes the biosynthesis and biodegradation of Fru-2,6-P2. The characterization of this system and of its regulation is a prerequisite for understanding of how fluctuations in Fru-2,6-P2 concentration integrate the hormonal and metabolic signals that control glycolysis. The first committed step of glycolysis is the conversion of fructose 6-phosphate (Fru-6-P) and ATP into fructose l,6-bisphosphate catalyzed by 6-phosphofructo-l-kinase (PFK-1). The most potent stimulator of PFK-1 is fructose 2,6-bisphosphate (Fru-2,6-P2), present in all eukaryotes. Unlike other regulatory ligands of PFK-1 that act at millimolar concentrations, Fru-2,6-P, stimulates PFK-1 as an allosteric effector active in the micromolar (physiological) range. It does so by increasing the affinity of PFK-1 for Fru-6-P and by relieving the inhibition caused by ATP. Thus, Fru-2,6-P2 exerts a strategic control over glucose utilization. The properties of this system were studied first in rat liver and are summarized in the chapter for comparison with those of other tissues.

Regulation of Bacillus subtilis gene expression during the transition from exponential growth to stationary phase.

Abstract: Publisher Summary This chapter discusses the current understanding of B. subtilis regulatory proteins. A number of regulatory proteins function to activate or modulate expression levels of many transition-state genes. The main teleological difference between these regulators and the transition-state regulators just discussed is that, while the latter are active during vegetative growth to silence post-exponential specific genes, the former primarily make their presence felt only after the onset of the transition state. Genetic and biochemical aspects of B. subtilis post-exponential gene expression have been studied intensively for some time because of their association with the developmental program of endospore formation. Therefore, the transition state of B. subtilis can be viewed as a crossroads, where functions necessary for smooth entry into any of the alternate paths are present, but before any final commitment has been made. The proper regulation of gene expression during the transition state is obviously critical for optimizing survival. In fact, in its natural soil habitat, where nutrients are usually limiting, the transition state is probably the predominant metabolically active state of a Bacillus cell.

Cell Delivery and Mechanisms of Action of Antisense Oligonucleotides

Abstract: Publisher Summary This chapter discusses the synthetic nucleic acids interfering with gene expression (SNAIGE), which are being developed as specific regulators of gene expression both for fundamental studies and/or for potential (medical) applications. Although straightforward in principle, the approach still faces many unknowns. Cell-free studies with model systems have helped one to learn much about the various modes through which oligonucleotides bind to their nucleic acids or protein targets. Increasing numbers of modifications allowing improved resistance toward nucleases or more efficient interaction with various targets have been devised and more will be forthcoming. A most exciting prospect has been enabled by synthetic material; the normal backbone of DNA has been replaced by a polyamide linkage, giving rise to “polyamide nucleic acid” that retains specificity in base recognition. Although a large number of genes have been successfully down-regulated in various biological systems. The tools of cell biology will help in solving pending questions, such as intracellular routing of SNAIGE, involvement of RNase H in their action, and accessibility of various sites in mRNAs, pre-mRNAs, or DNAs in their cellular environment. This helps to devise new tools on a more rational basis and to choose the best strategy in each case.

Bacterial adenylyl cyclases.

Abstract: Publisher Summary This chapter describes the enzyme (adenylyl cyclase) that effect the synthesis of Adenosine 3’,5’-cyclic monophosphate (cAMP) in various bacterial species. The content will rather reflect current major interests. The adenylyl cyclase from Escherichia coli has been a major subject of research interest ever since it was identified as the probable point for physiological regulation of cAMP levels in that organism and therefore a prime candidate for a protein mediator of the catabolite repression response mechanism. cAMP functions as a cytoplasmic element mediating some reactions crucial for efficient cellular function. An activity that has been found only in eukaryotic cells is the CAMP-dependent protein kinase. This enzyme is a well-known target of the action of cAMP as a second messenger, in which action this ligand transmits a signal generated by an extracellular hormone. The manner in which cAMP acts on the cAMP-dependent protein kinase involves a release of the catalytic moiety of the enzyme from a complex in which its activity is inhibited as a result of binding to a regulatory subunit.

Immunoglobulin gene diversification by gene conversion.

Abstract: Publisher Summary This chapter discusses recent advances in the study of gene conversion, primarily in the chicken, and on current models for the molecular mechanism of this form of somatic gene conversion. Most of the higher vertebrates, including teleost fish, the amphibian Xenopus, the reptile Caiman, and most of the mammalian species studied, appear to rely on these combinatorial and junctional mechanisms to generate the primary antibody repertoire. Gene conversion in the chicken immunoglobulin light chain (IgL) locus has been shown to be restricted to the rearranged V gene segment, and does not occur in the leader region, the J gene segment, or within a germline IgL allele that has not undergone V-J joining. The chicken immunoglobulin heavy chain IgH and IgL loci are novel in that, they each contain only single V and J gene segments that are capable of undergoing V(D)J joining in B cell progenitors. In addition, V(D)J recombination in the chicken is not an ongoing development process. V(D)J joining is complete by day 18 of embryogenesis, and chicken progenitor B cells then migrate to a specialized lymphoid organ, the bursa of Fabricius, which is a posterior invagination of the cloaca of avian species. More recently, gene conversion has also been demonstrated to generate somatic immunoglobulin diversity in rabbit, a mammalian species well known for the diversity of its immune response. Although less is known about the mechanism of gene conversion than that of V(D)J joining, recent work has begun to shed some light on the molecular mechanisms involved in immunoglobulin gene conversion.

Enzyme organization in DNA precursor biosynthesis.

Abstract: Publisher Summary This chapter discusses the process by which DNA precursors are synthesized and delivered to replication sites and consider the possibility of physical or functional linkage between enzymes of deoxyribonucleoside 5‘-triphosphate (dNTP)’ synthesis and those directly involved in the replication DNA replication has been shown in both prokaryotic and eukaryotic systems to involve the cooperative function of numerous proteins associated at replication forks Many metabolic pathways involve multifunctional enzymes or tightly associated multienzyme complexes Examples of the former are the fattyacyl- CoA synthetase enzyme in vertebrate cells and the eukaryotic “CAD protein” and UMP synthetase, which catalyze the first three and last two reactions, respectively, of the six steps in de novo pyrimidine-nucleotide synthesis Examples of tightly but non-covalently bound aggregates include the well-known pyruvate and α-ketoglutarate dehydrogenase complexes of mitochondria The potential biological advantages of enzyme association are fairly obvious and might include: maintenance of locally high metabolite concentrations without the need for physical compartmentation within a membrane; protection of the solvation capacity of cell water, because average concentrations of most metabolites are kept low, even though local concentrations may be much higher; efficiency of regulation of both enzyme synthesis and metabolic fluxes; and relative rapidity with which a metabolic process can respond to changes in the intracellular environment

Identification and characterization of novel substrates for protein tyrosine kinases.

Abstract: Publisher Summary This chapter discusses: (1) the strategies used to identify Protein tyrosine kinase (PTK) substrates and (2) the recent progress made in identifying new and interesting cytoskeletal protein components that serve as substrates for oncogenic PTKs. PTKS play an important role in the transmission of signals for a broad spectrum of cellular activities, including the activation of secretory events, T-cell and B-cell activation, responses to cell-extracellular matrix interactions, differentiation, mitogenesis, and oncogenesis. Because of the desire to understand the molecular basis of carcinogenesis, the roles played by PTKs in mitogenesis and oncogenesis have been most extensively studied. PTKs have also been identified as the translation products of a number of oncogenes. Expression of these tyrosine kinase oncoproteins in cultured cells results in pronounced alterations in cell shape and growth control, and in many cases, renders the transformed cells tumorigenic in syngeneic animals or nude mice. In addition, inoculation of retroviruses containing a tyrosine kinase oncogene into animals often leads to the rapid induction of leukemias or solid tumors. Thus, oncogenic PTKs subvert the regulation of normal cell growth, presumably by chronically transmitting a mitogenic signal into the cell.

Posttranscriptional Control of the Lysogenic Pathway in Bacteriophage Lambda

Abstract: Publisher Summary This chapter discusses the recent developments in the study of Lambda (λ) phage gene expression showing that post-transcriptional control of phage genes by host functions provides highly sensitive regulatory circuits. The control of transcription initiation from the early promoters of bacteriophage λ is not sufficient to provide for subtle controls involved with the switch between lysogenic and lytic pathways. The ability of λ to choose between lytic and lysogenic development has evolved as a response to changes in the physiological state of the host cell. The frequency by which the phage enters the lytic or lysogenic pathway is determined in large measure by the nutritional state of the host cell. For example, starved cells lysogenize more efficiently than cells grown in a rich medium. In addition, the number of infecting phage particles per cell is also known to determine the rate of lysogenization: the higher the multiplicity of infection, the higher the rate of lysogenization. It is probable that the physiological state of the cell is signaled to the phage by host regulatory factors.

Adenoviral DNA Integration and Changes in DNA Methylation Patterns: A Different View of Insertional Mutagenesis

Abstract: Publisher Summary Adenovirus DNA integration in many respects is akin to non-homologous recombination. Nevertheless, “patchy” homologies at the sites of integration have frequently, though not invariably, been found. Perhaps, the terms “homologous” and “non-homologous recombination” are still too abstract to allow an adequate description of an obviously very versatile cellular mechanism. Furthermore, transcriptionally active regions of the cellular genome have a higher propensity to recombine with foreign DNA, and one can argue plausibly that such regions may present the right chromatin structure for the recombination with foreign DNA. Moreover, foreign genes could gain functional advantages by insertion into chromatin structures that are in the process of active transcription. Moreover, insertion can lead to local mutagenesis. Researchers on gene therapy, who must be concerned with the functionally competent insertion of foreign genes into a pre-existing genome, will be preoccupied with the mechanism of insertion and with the problems of how to keep inserted genes active or dependent on controllable regulatory mechanisms.

DNA Polymerase II, the Epsilon Polymerase of Saccharomyces cerevisiae

Abstract: Publisher Summary This chapter discusses the data on DNA polymerase II in the context of current knowledge of eukaryotic DNA polymerases, DNA replication and its fidelity, and DNA repair. DNA polymerase II has been proffered as the “repair polymerase.” Contrary to the implicit assumption, DNA repair is not a single process but an array of different pathways that have been dissected at least partially by genetic studies. It cannot be presumed that a particular polymerase acts in repair but not in replication, or that a single polymerase performs all DNA repair, or that each repair pathway has a dedicated polymerase. If DNA polymerase II is involved in the repair of damaged DNA, it might be possible in principle to obtain radiation-sensitive mutants either in POL2 , or in DPB2, or DPB3. Further evidence implicating DNA polymerase II in DNA replication comes from the spontaneous mutator phenotype of the exonuclease-deficient pol2-4 mutant. While a spontaneous mutator phenotype may also arise in DNA repair mutants, the pol2-4 mutation does not confer sensitivity to DNA-damaging agents, and the epistatic relationships discussed in the chapter, link the DNA polymerase II 3’→5’ exonuclease with DNA replication.

Genomic Organization of T and W, a New Family of Double-Stranded RNAs from Saccharomyces cerevisiae

Abstract: Publisher Summary This chapter describes the characterization of two dsRNAs. Most of the information comes from cloning and sequencing of their cDNAs. Their nucleotide sequences reveal that they are of viral origin, but their non-encapsidated nature distinguishes them from other dsRNA viruses. T and W were first described as dsRNAs present in a number of different laboratory strains of S. cerevisiae; the sizes of T and W were estimated to be 2.7 and 2.25 kb, respectively, in agarose gels. Both T and W are cytoplasmically inherited and are not encapsidated into viral particles. Both are inducible in certain yeast strains when grown at 37°C. W is present in most laboratory yeast strains. The fact that T has always been found in W-carrying strains may suggest that T is dependent on W for its maintenance. Therefore, T and W dsRNAs are different from the other dsRNAs present in S. cerevisiae and they are maintained autonomously in the yeast cells, probably by an RNA-to-RNA replication pathway.

Enzymology of homologous recombination in Saccharomyces cerevisiae.

Abstract: Publisher Summary This chapter discusses the current state of knowledge of the proteins that function in genetic recombination in the simple eukaryote Saccharomyces cerevisiae To provide a framework, a brief discussion of current views of the molecular mechanism(s) of genetic recombination, particularly recombination models, is discusses in the chapter Genetic experiments that predict DNA structures involved in the recombination have led to the proposal of molecular models for recombination Physical characterizations of recombination intermediates that occur in vivo have provided support for proposed recombination models Mutational analysis has identified gene products required for the recombination, and has defined recombination pathways and mechanisms The availability of such mutants has provided a means of identifying, overproducing, and purifying recombination proteins Enzymatic studies have provided in vitro recombination systems for identifying and characterizing recombination proteins With the introduction of cytology in studying recombination in the yeast, S cerevisiae, another indicator for a possible function of proteins encoded by genes controlling recombination has been developed The precise subcellular localization of proteins, as well as the effect of mutations on chromosome structure may reveal a role in recombination For example, mutations in the HOP1 gene of S cerevisiae abolish recombination between homologs (interchromosomal recombination) but HOP1 mutant are proficient in intrachromosomal recombination between duplicated sequences

ADP-ribosylation factors: protein activators of cholera toxin

Abstract: Publisher Summary The toxins alter the activity of the guanine nucleotide-binding proteins (G proteins) by catalyzing the transfer of the ADP-ribose moiety of NAD to a critical amino acid in the a subunit. This ADP-ribosylation reaction results in either activation (e.g., cholera toxin, E. coli heat-labile enterotoxin) or inactivation (e.g., pertussis toxin) of the G protein. The G proteins are heterotrimeric signal-transducing proteins responsible for coupling cell-surface receptors to their intracellular effectors. Cholera toxin and E. coli heat-labile enterotoxins catalyze the ADP-ribosylation of the α - subunit of G s , the stimulatory G protein of the adenylylcyclase system that is also believed to regulate Ca 2+ channels. ADP-ribosylation by pertussis toxin of the G αi, G αo, and G αt proteins on a cysteine residue near the carboxy terminus interferes with their ability to interact functionally with cell-surface receptors, thereby interrupting transmembrane signalling. In addition to these proteins, there is a family of G α subunits that appear not to be substrates for any of these bacterial toxins.

Analysis of Rice Genes in Transgenic Plants

Abstract: Publisher Summary This chapter discusses the information on molecular analyses of rice genes by in vivo and in vitro methods and in transgenic plants. It selected examples of several important types of rice genes, particularly those about which ones have first-hand information. Another consideration in choosing specific genes for discussion involves selection of those genes that are unique to plants: for example, genes related to seed storage proteins in rice and other monocotyledonous plants, such as wheat and barley. The response of rice phytochrome genes to light is another such example of gene regulation unique to plants. However, the development of specific applications, such as attempts to produce insectresistant transgenic rice plants, is a unique and important application of plant biotechnology. Moreover, in plant research, work on dicotyledonous plants such as tobacco and Arabidopsis is more advanced than that on monocotyledonous plants, such as rice and wheat because these dicots are easier to transform and regenerate more plants than monocots.