Showing papers in "Progress in Nucleic Acid Research and Molecular Biology in 1991"

Vertebrate protamine genes and the histone-to-protamine replacement reaction.

Abstract: Publisher Summary This chapter discusses Bloch's classes Type 1 (“true” protamines) and Type 2 (“stable” protamines), for which substantial molecular information is now available. Protamines are defined as having a cysteine-plus-arginine composition of 45–80 mol% and a serine plus- threonine content of 10–25 mol%. However, it should be mentioned that an additional class of sperm protamines found in the sperm nuclei of certain flat fish in the order Pleuronectiformes. These protamines are rich in arginine and serine, but are very large (80,000–200,000 daltons) compared to the typical true and stable protamines ranging from 5000 to 10,000 Da. In addition, the fact that distinctive amino acids, such as threonine or valine, present in chicken protamine polypeptides, are conserved in position in the amino acid sequence of quail only if the sequence redetermined by Oliva and Dixon is considered, makes the probability of different allelic variants in chicken much less likely.

Ribosome Biogenesis in Yeast

Abstract: Publisher Summary This chapter focuses on the concept of ribosome biogenesis in yeast. The formation of functional ribosomes is a highly complex phenomenon requiring the interplay of a large number of molecular processes. Ribosomes contain, depending on their origin, some 60–80 different components, proteins, and RNA molecules, most in a single copy per ribosome. Because normally growing cells generally contain no free pools of these ribosomal components, the expression of the numerous ribosomal genes must be subject to tight coordinate control to ensure the production of equimolar amounts of the various rRNA and ribosomal-protein (r-protein) constituents. Because of its accessibility to genetic and physiological manipulation, the yeast— Saccharomyces cereuisiae— has become one of the most popular organisms for studying the questions raised by the coordinate control of ribosome biogenesis in eukaryotes. The genes for the various yeast rRNAs and those for about 40 of the yeast r-proteins have been cloned and subjected to studies. As a result, the amount of information collected on the structure and function of ribosomal genes in yeast surpasses that available for any other eukaryote. This information has provided important insights into the regulatory mechanisms controlling the expression of these genes. In addition, progress has been made in developing systems that provide access to the hitherto largely unexplored field of eukaryotic ribosome assembly, including the mechanism of rRNA processing.

Aminoacyl-tRNA Synthetase Family from Prokaryotes and Eukaryotes: Structural Domains and Their Implications

Abstract: Publisher Summary This chapter discusses on the evolutionary acquisitions in order to propose a comprehensive view concerning the possible physiological implications of the chain extensions that characterized the lower as well as higher eukaryotic aminoacyl-tRNA synthetases, as compared to their prokaryotic counterparts. Aminoacyl-tRNA synthetases are ubiquitous enzymes that play a key role in protein synthesis. This family of 20 enzymes offers a suitable tool for the study of structure/function relationships for a class of proteins involving similar functions, yet with different substrate specificities. Aminoacyl-tRNA synthetases are essential enzymes encountered in all living cells. They catalyze the esterification of one amino acid to the 3’ end of the corresponding tRNA. From an evolutionary point of view, the knowledge of primary structures for numerous aminoacyl-tRNA synthetases, including those for 18 enzymes from prokaryotes, has pointed out some common structural features arguing in favor of an extensive relationship among these enzymes. However, molecular and biochemical studies on eukaryotic aminoacyl-tRNA synthetases have delineated distinctive features acquired during evolution.

Nucleosome positioning: occurrence, mechanisms, and functional consequences.

Abstract: Publisher Summary This chapter describes the salient features of chromatin structure involved in positioning, reviews examples of non-random location of nucleosomes, examines mechanisms that have been proposed for positioning and experimental tests of several of these, and discusses some recent efforts to ascertain whether positioning could and/or does have any effect on the function of DNA in chromatin. There is no experimental evidence for, and sound theoretical arguments against, positioning of nucleosomes for most of any eukaryotic genome. However, strong evidence is accumulating for positioning of some nucleosomes. Almost any mechanism that has been postulated for the positioning has found experimental support in some system. It seems clear that there will be hierarchies in the strength of positioning signals and mechanisms that may lead to variability in the location of nucleosomes in different cell types. However, a positioned nucleosome is located in a precise site relative to DNA sequence in all cells of a given population. In this situation, any particular DNA sequence in the region of the positioned nucleosome would always lie in the same relationship to histones-in the linker or in the core particle, in the central or the peripheral region of the core particle, etc.

Genetics of Human Alcohol-Metabolizing Enzymes

Abstract: Publisher Summary This chapter explains the molecular genetics of human alcohol dehydrogenase and aldehyde dehydrogenase. Relationships between the genetic variations and alcohol-related physiological problems have become apparent. More than 80% of ethanol administered is oxidized by alcohol dehydrogenase and most of the acetaldehyde thus formed is further oxidized to acetate by aldehyde dehydrogenase in the liver. Alcohol dehydrogenases are enzymes catalyzing the conversion of various alcohols to the corresponding aldehydes by means of an NAD+-dependent oxidation. The human ADH isozyme system is extremely complex and varied among individuals. Based on the analyses of electrophoretic variations and the mode of inheritance of isozyme patterns, it was proposed that: three non-allelic loci are involved in the synthesis of three types of subunits, α, β, and γ, respectively. The ADH2 and ADH3 loci are dimorphic— that is, there are two common alleles, ADH12 and ADH22, and ADH31 and ADH23 respectively, in each locus; and the catalytically active ADH isozymes are homo- and heterodimers of these wild-type and variant-type subunits. Later, an additional common variant allele in the ADH2 locus was found in American blacks. The model proposed has been confirmed by cloning and by characterization of cDNAs and genes.

Specific Interaction between RNA Phage Coat Proteins and RNA

Abstract: Publisher Summary This chapter describes the biochemistry of the interaction of phage coat protein with RNA and attempt to provide a molecular understanding of its high specificity. Coat protein binding is believed to serve two functions in the life cycle of the phage: 1) it acts as a translational repressor of the replicase gene early in infection, and 2) as an initiation site of phage assembly late in infection. This interaction has been extensively a prototype of sequence specific RNA-protein interactions. It is now clear that the phage coat proteins can be considered an example of a class of RNA hairpin binding proteins that are quite common in prokaryotes and eukaryotes. In case of bacteriophage coat protein, the coat protein assembles into phage-like capsids that can be purified by differential centrifugation and ion-exchange chromatography. Most coat proteins can be successfully renatured by the transfer from storage buffer directly into a variety of neutral buffers of moderate ionic strength. In many cases, these renatured proteins are fully active in both RNA binding and capsid assembly.

Recognition of tRNAs by aminoacyl-tRNA synthetases.

Abstract: Publisher Summary This chapter describes the recognition of tRNAs by aminoacyl-tRNA synthetases. The highly specific selection of tRNA substrates by aminoacyl-tRNA synthetases is an intriguing problem in RNA-protein recognition. Synthetases specific for each of the 20 amino acids encounter a pool of tRNAs in the cell having similar overall structures. The selection of the appropriate tRNAs for the attachment of each amino acid occurs by the formation of RNA-protein contacts unique to each cognate tRNA-synthetase pair. The sites in tRNAs that govern these interactions have been investigated by a variety of techniques discussed in the chapter, such as the assays of the amino-acid-acceptor specificity of tRNA. The role of the anticodon in recognition, role of the acceptor Stem and discriminator base, and role of modified bases is discussed in the chapter. The anticodon is not required for the recognition of tRNA AIa , and possibly tRNA Ser ; these and all other E. coli tRNAs probably contain identity elements in the anticodon that are crucial for the discrimination of cognate and noncognate tRNAs by synthetases in vivo. In addition, a number of E. coli tRNAs contain important recognition elements for their cognate synthetases in the acceptor stem and/or at the discriminator site.

Structural elements in RNA.

Abstract: Publisher Summary This chapter describes the RNA structural characteristics that have emerged so far. Folded RNA molecules are stabilized by a variety of interactions, the most prevalent of which are stacking and hydrogen bonding between bases. Many interactions among backbone atoms also occur in the structure of tRNA, although they are often ignored when considering RNA structure because they are not as well-characterized as interactions among bases. Backbone interactions include hydrogen bonding and the stacking of sugar or phosphate groups with bases or with other sugar and phosphate groups. The interactions found in a three-dimensional RNA structure can be divided into two categories: secondary interactions and tertiary interactions. This division is useful for several reasons. Secondary structures are routinely determined by a combination of techniques discussed in chapter , whereas tertiary interactions are more difficult to determine. Computer algorithms that generate RNA structures can search completely through possible secondary structures, but the inclusion of tertiary interactions makes a complete search of possible structures impractical for RNA molecules even as small as tRNA. The division of RNA structure into building blocks consisting of secondary or tertiary interactions makes it easier to describe RNA structures. In those cases in which RNA studies are incomplete, the studies of DNA are described with the rationalization that RNA structures may be analogous to DNA structures, or that the techniques used to study DNA could be applied to the analogous RNA structures. The chapter focuses on the aspects of RNA structure that affect the three-dimensional shape of RNA and that affect its ability to interact with other molecules.

Lens proteins and their genes.

Abstract: Publisher Summary Recent developments made in lens research have demonstrated that certain enzyme proteins occurring in nonlenticular tissues in minor quantities exist in the lens in high concentration and seem, therefore, to be recruited as structural elements in evolution. In addition, it appears that at least, one of the typical structural proteins of the vertebrate lens—αB-crystallin—is by no means lens-specific. For example, heart, brain, skeletal muscle, kidney, and possibly other tissues express this characteristic component of the αcrystallin aggregate. In addition, research community is beginning to understand the way genes are expressed and regulated in the eye lens. Studies using gene constructs containing crystallin 5’-regulating sequences have enabled the mapping of positive and negative control elements responsible for tissue-specific expression. However, many features, such as post-transcriptional modifications, still have to be clarified at this level.

DNA helicases of Escherichia coli.

Abstract: A great deal has been learned in the last 15 years with regard to how helicase enzymes participate in DNA metabolism and how they interact with their DNA substrates. However, many questions remain unanswered. Of critical importance is an understanding of how NTP hydrolysis and hydrogen-bond disruption are coupled. Several models exist and are being tested; none has been proven. In addition, an understanding of how a helicase disrupts the hydrogen bonds holding duplex DNA together is lacking. Recently, helicase enzymes that unwind duplex RNA and DNA.RNA hybrids have been described. In some cases, these are old enzymes with new activities. In other cases, these are new enzymes only recently discovered. The significance of these reactions in the cell remains to be clarified. However, with the availability of significant amounts of these enzymes in a highly purified state, and mutant alleles in most of the genes encoding them, the answers to these questions should be forthcoming. The variety of helicases found in E. coli, and the myriad processes these enzymes are involved in, were perhaps unexpected. It seems likely that an equally large number of helicases will be discovered in eukaryotic cells. In fact, several helicases have been identified and purified from eukaryotic sources ranging from viruses to mouse cells (4-13, 227-234). Many of these helicases have been suggested to have roles in DNA replication, although this remains to be shown conclusively. Helicases with roles in DNA repair, recombination, and other aspects of DNA metabolism are likely to be forthcoming as we learn more about these processes in eukaryotic cells.

Nuclear RNA-binding proteins.

Abstract: Publisher Summary The control of gene expression involves several steps at which specific sequences in pre-mRNA transcripts, as well as those in small RNA molecules, are recognized by proteins. RNA-binding proteins can be expected to mediate interactions in a variety of cellular processes, including those occurring in the transcription complex, the spliceosome and ribosome. The members of one family of nuclear proteins that bind to RNA contain a specific RNA recognition motif (RRM). The RRM family of proteins functions at several levels in RNA processing and some family members are involved in tissue-specific and developmentally regulated gene expression. This chapter describes the proteins that contain this RRM and discusses the potential involvement of these proteins in the control of gene expression at the level of RNA processing. These proteins are modular in structure and often contain at least two types of interactive surfaces—one or more that interacts specifically with RNA and another that interacts with other molecules. Studies to date indicate that despite the strong homology among these proteins, they have unique properties of recognition that allow them to distinguish the RNAs of diverse structure.

Amplification of DNA sequences in mammalian cells.

Abstract: Publisher Summary This chapter discusses the aberrant DNA sequence amplification processes that occur in mammalian cells because of the clinical relevance to drug resistance and oncogene amplification in tumors and because the underlying mechanisms seem close to being understood at the molecular level. What seemed to be a relatively esoteric mutational phenomenon is now a major determining factor in the genesis of cancer, as well as a serious deterrent to successful chemotherapeutic drug treatment regimens. The chapter traces the history of the discovery of DNA sequence amplification, cites several examples, and discusses the similarities and differences among these systems. Important methodologies developed for studying amplified sequences are also presented in the chapter. It reviews the viable models that attempt to explain the phenomenon in mammalian cells and shows the way the resolution of the underlying molecular mechanisms of amplification promises to teach much about both normal and aberrant chromosome dynamics (e. g., replication, recombination and repair processes, and chromosomal breakage and healing).

msDNA of bacteria.

Abstract: The msDNA-retron element represents the first prokaryotic member of the large and diverse retroelement family found in many eukaryotic genomes (Table II). This prokaryotic retroelement exists as a single copy element in the chromosome of two different bacterial groups: the common soil microbe M. xanthus and the enteric bacterium E. coli. It encodes an RT similar to the polymerases found in retroviruses, containing most of the strictly conserved amino acids found in all RTs. The RT is responsible for the production of an unusual extrachromosomal RNA-DNA molecule known as msDNA. Each composed of a short single strand of RNA and a short single strand of DNA, msDNAs vary considerably in their primary nucleotide sequences, but all share certain secondary structural features, including the unique 2',5' branch linkage that joins the 5' end of the DNA chain to the 2' position of an internal guanosine residue of the RNA strand. It is proposed that msDNA is synthesized by reverse transcription of a precursor RNA transcribed from a region of the retron containing the genes msr (encoding the RNA portion) and msd (encoding the DNA portion) and the ORF (encoding the RT). The precursor RNA transcript folds into a stable secondary structure that serves as both the primer and the template for the synthesis of msDNA. The msDNA-retron elements of E. coli are found in less than 10% of all strains observed, are heterogeneous in nature, and have an atypical aminoacid codon usage for this species, suggesting that this element was transmitted to E. coli by some other source. The presence of directly repeated 26-base-pair sequences flanking the junctions of the Ec67-retron of E. coli also suggests that it may be a mobile element. However, the msDNA-retrons of M. xanthus appear to be as old as other genes native to this species, based on codon-usage data for the RT genes and the fact that every strain of M. xanthus appears to have the same type of msDNA. If the msDNA-retron element originated with the myxobacteria, it would place the existence of retrons before the appearance of eukaryotic cells, suggesting that the bacterial element is perhaps the ancestral gene from which eukaryotic retroviruses and other retroelements evolved.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular structure and transcriptional regulation of the salivary gland proline-rich protein multigene families.

Abstract: Publisher Summary This chapter focuses on the biochemistry and molecular biology of salivary proline-rich proteins (PRPs). Specialized cells in eukaryotes variably express different genes during differentiation and development. Exocrine glands, such as the pancreas and salivary glands, have served as the models of secretory tissues. Under ordinary conditions, the salivary glands of adult animals are relative stable and do not change appreciably in cell size or number. However, the administration of catecholamine isoproterenol causes dramatic morphological, cytological, and biochemical changes. Morphologically, the parotid glands can increase up to 10-fold in size. Cytologically, about 50% of the acinar cells are polyploid within 2 days of treatment. Biochemically, a dramatic induction of the multigene family encoding the PRPs is observed. The expression of PRPs for the parotid and submandibular glands is tissue-specific or, possibly more correctly, cell-specific. PRPs have been identified immunochemically in the trachea and pancreas, but there is no evidence that PRP genes in these tissues respond as in the salivary glands to isoproterenol treatment. Small amounts of PRP mRNAs are observed in the mouse pancreas after isoproterenol treatment, but these results are variable. Current data suggest that transcriptional controls, tissue-specific factors, and post-translational modification share the role of principal modulators of the expression of the PRP gene families.

Molecular-biology approaches to genetic defects of the mammalian nervous system

Abstract: Publisher Summary This chapter discusses the molecular-biology approaches to genetic defects of the mammalian nervous system. One effective means for studying brain molecular function is the isolation and characterization of genes encoding neuroactive substances, their synthetic enzymes and receptors, and molecules that share sequence relationships with these proteins. These studies amount to a direct extension of classical biochemical analysis and represent a major endeavor of modern molecular neurobiology. A limitation of this research is that it can only produce information about the molecules for which there are already known functions, and hence assays. It is, thus, uninformative about a considerable portion of neural molecules for which functions have not already been postulated. Two alternative strategies that can be informative about novel brain proteins use complementary approaches. One involves the isolation and characterization of genes defined by neurological mutations, allowing direct correlations to be made between mutant neural phenotypes and protein structure. The second is the study of genes whose products are restricted to neural tissues, but whose functions are unknown. Recent advances in technology indicate that these approaches can be potentially as illuminating as more traditional studies on known brain proteins.