RNase protection

1 Apr

RNase protection

nomenon was discovered in the mitochondria of try- panosomes, and the edited molecules allowed trans- lation of functional proteins in situations where the unedited mRNAs were defective. In trypanosomes the mtDNA is made up of 25-50 maxicircles (each 20-40 kilobases) and hundreds of minicircles (each 1-3 kilobases). The maxicircles contain the mito- chondrial genes, while the minicircles encode guide RNAs, each about 40 nucleotides long. The gRNAs bind to specific sites on the mRNAs and subse- quently insert or delete uridines. See Appendix C, 1986, Benne et al.; 1990, Blum, Bakalara and Simp- son; mitochondrial DNA, proofreading, Trypanosoma. RNA gene a DNA segment coding for one of the various types of nonmessenger RNA (rRNA, 5S RNA, or tRNA). RNA interference (RNAi) a cellular process in which endogenous or exogenous double-stranded RNA (dsRNA) (q.v.) molecules induce gene silenc- ing by mediating the degradation of target messen- ger RNAs (mRNAs), or by other regulatory means. In this process, dsRNA precursor molecules are pro- cessed into double-stranded small interfering RNAs (siRNAs) (q.v.), approximately 22 nucleotides long, by the enzyme Dicer (q.v.). The siRNA duplex then associates with an endonuclease-containing, multi- protein complex, known as RNA-induced silencing complex (RISC) and unwinds in an ATP-dependent manner.

The activated RISC and its associated siRNA subsequently bind complementary sequences in the target mRNA, and the mRNA is cleaved and destroyed. RNAi has some intriguing features: gene silencing can spread from one tissue to the entire or- ganism, and it can also be transmitted through the germ line for some generations. RNAi occurs natu- rally in a variety of organisms and is thought to play a role in regulating gene expression and in protecting organisms from viruses and transposable elements. It can also be induced experimentally as a tool for cre- ating loss of function phenotypes (e.g., by transfect- ing cells or embryos with chemically synthesized siRNAs). In Caenorhabditis elegans RNAi has been used to analyze the function of all genes residing on a specific chromosome. See Appendix C, 1998, Fire et al.; 2000, Fraser et al.; RNA transfection. RNA ligase an enzyme, such as T4 RNA ligase (q.v.), that can join RNA molecules together.

See Appendix C, 1972, Silber et al. RNA-P I, II, III See RNA polymerase. RNA phage an RNA bacteriophage such as MS2 and Q beta. RNA polymerase (RNA-P) an enzyme that tran- scribes an RNA molecule from the template strand of a DNA molecule. Two kinds of RNA polymerases are known in prokaryotes: one produces the RNA primer required for DNA replication; the other tran- scribes all three types of RNA (mRNA, tRNA, rRNA). In eukaryotes, each type of RNA is tran- scribed by a different RNA polymerase. RNA poly- merase I (RNA-PI) resides in the nucleolus and cata- lyzes the synthesis of rRNA. RNA-PII is localized in the nucleoplasm, where it catalyzes the synthesis of mRNA. RNA-PII is specifically inhibited by alpha amanitin (q.v.). RNA-PIII makes tRNA, 5S RNA, and other small RNA molecules. The structure of the RNA polymerase of E. coli is known in great detail. It has the general formula ωαασβ′β. The component proteins, their relative molecular masses, the encoding genes, and their locations are shown here.

The catalytic site for RNA polymerization is thought to reside in the beta protein, and this is also the place where rifampicin (q.v.) binds. The sigma pro- tein functions in promoter recognition and the initia- tion of RNA synthesis. Eukaryotic RNA polymerases are still more complex, since they contain about 10 rather than five types of subunit. See Appendix C, 1961, Weiss and Nakamoto; 1969, Roeder and Rut- ter, Burgess et al.; amatoxins, Cajal body, Miller trees, muscular dystrophy, nucleolus, nucleolus organizer, polynucleotide, phosphorylase, ribosomal RNA genes, strand terminologies, TATA-box binding protein, ter- minator, T7 RNA polymerase, transcription unit.

RNA primer  See primer RNA.
RNA processing  See posttranscriptional processing.
RNA puff  See chromosomal puff.
RNA replicase  an RNA-dependent RNA polymer-ase (q.v.). See MS2 and Q beta.
RNase  any enzyme hydrolyzing RNA.

RNase  protection  a  technique  for  locating  the points of effective contact between a nucleic acid chain and a cognate polypeptide chain; the complex (e.g., tRNA and its cognate aminoacyl-tRNA synthe- tase) is treated with a group of RNases that digest

RNA splicing

all of the RNA except those regions in contact with the synthetase. See photoactivated cross-linking. RNA splicing the removal of noncoding regions from a large precursor RNA molecule, and the nu- cleotide sequences transcribed from nonadjacent DNA segments are then joined together to produce a smaller mature RNA. See alternative splicing, in- tron, posttranscriptional processing, splice junctions, spliceosome. RNA transfection the experimental introduction of exogenous single- or double-stranded RNA (ds RNA) molecules into a cell or an embryo, which re- sults in a phenotypic change that is transient in na- ture (i.e., is not stably inherited). Transfection of cells with a specific messenger RNA (mRNA) (q.v.) can be used to express a particular protein, whereas transfection with certain single- or ds RNA mole- cules can produce RNA interference (q.v.).

RNA transfection can be achieved through a number of methods, including microinjection (q.v.), electropor- ation (q.v.), the use of viral vectors, and the use of liposomes (q.v.). Furthermore, in C. elegans, RNA transfection (and resulting RNA interference) can be achieved by soaking the animals with ds RNA or feeding them E. coli cells which express ds RNA. RNA vector a retrovirus used to transfer a provirus carrying a beneficial foreign gene into the chromo- some of a host. An example would be using a geneti- cally engineered HIV which contains a functioning beta globin gene to infect a mouse genetically engi- neered to have a beta globin deficiency. See Appen- dix C, 2001, Pawliuk et al. RNP ribonucleoprotein (q.v.). rNTP ribonucleoside 5′-triphosphates. Robertsonian translocation See centric fusion. rod one of the elongate, unicellular photoreceptors in the vertebrate retina (q.v.), involved with vision in dim light.

Rods do not discriminate color differ- ences. See rhodopsin. Roentgen (R) the quantity of ionizing radiation that liberates 2.083 × 109 ion pairs in a cubic centi- meter of air (at 0°C and at a pressure of 760 milli- meters of mercury) or approximately two ion pairs per cubic micron of a substance such as protein (which has a density of 1.35).

A gram of tissue ex- posed to 1 roentgen of gamma rays absorbs about 93 ergs. The unit is named in honor of W. C. Roentgen, the discoverer of x-rays, who received the Nobel Prize in physics in 1901 for this work. roentgen equivalent physical (rep) the amount of ionizing radiation that will result in the absorp- tion in tissue of 93 ergs per gram. rogue a variation from the standard variety, usu- ally inferior. rolling circle a model mechanism for the replica- tion of DNA molecules, so named because the growing point can be imagined as rolling around a circular template strand. The circular DNA is shown here in A. In B, a nick opens one strand, and the free 3′-OH end is extended by DNA polymerase. The newly synthesized strand displaces the original pa- rental strand as it grows (C, D).

By E, the polymer- ase has completed one revolution, and by F, two rev- olutions. The result is a molecule containing three unit genomes, one old and two new. The displaced strand can then serve as a template for a comple- mentary strand. This mechanism is used to generate concatemeric duplex molecules (e.g., phage lambda, amplified rDNA in amphibian oocytes, etc.). This type of DNA replication is sometimes called sigma replication because the structure produced by the rolling circle resembles the Greek lower case sigma (σ). The rolling circle mechanism also occurs in viro- ids (q.v.). Here the infectious (+) viroid RNA serves as a template for an RNA polymerase, which gener- ates a concatomeric, complementary (−) strand. In turn, this serves as template for synthesis of a (+) concatomeric RNA, which is later cleaved into geno- mic units that are then ligated to form circles. See Appendix C, 1968, Gilbert and Dressler; hammer- head ribosome, plus (+) and minus (−) viral strands, theta replication.

rolling-circle transposon a helitron (q.v.). Romalea microptera the lubber grasshopper. Mei- osis has been extensively studied in this species. Romanov the ruling family of Russia for almost 300 years. The last Tsar, Nicholas II, and his wife, Alexandra, had five children of which the first four were girls (Olga, Tatiana, Maria, and Anastasia). Then came Alexei, the male heir to the throne. Un- fortunately he inherited hemophilia (q.v.) from his

R (resistance) plasmid
grandmother, Victoria, Queen of England. As a re- sult he was in poor health during his short life. After the Revolution the Romanovs were imprisoned in the Ipatiev House at Yekaterinberg, Siberia. On July 16,1918, the entire family, their doctor, and three servants were murdered by a Bolshevik firing squad. In 1991 a grave was discovered containing the frag- mented remains of nine skeletons. Tests of mtDNA showed that five of the bodies were related. Subse- quent matches with the mtDNA of surviving rela- tives identified Nicholas and Alexandra. The Tsar’s mtDNA was found to be heteroplasmic (q.v.). The bodies of 14-year-old Alexei and his sister Maria were never found. See Appendix C, 1994, Gill et al.; mitochondrial DNA (mtNDA). root cap a cap of cells covering the apex of the growing point of a root and protecting it as it is forced through soil. root hair a tubular outgrowth of an epidermal cell of a root which functions to absorb water and nutri- ents from the soil. root nodules a small swelling on roots of legumes (q.v.) produced as a result of infection by symbiotic nitrogen-fixing bacteria.

See Rhizobium. Rosa the genus that includes the rose species, which have been extensively hybridized. Commer- cially grown species include R. centifolia, R. damas- cena, and R. multiflora. Rose chamber a closed culture vessel permitting long-term observation of explanted cells under phase microscopy. The fluid culture medium may be periodically renewed without disturbing the growing cells. R0t the product of RNA concentration and the time of incubation in an RNA-driven hybridization; the analog of C0t values used to describe DNA- driven hybridization reactions. rotational base substitution a break is induced by radiation at a corresponding point in both comple- mentary strands of a DNA molecule. The bond bro- ken is between the base and the sugar molecule to which it is attached.

Thus two complementary bases (held together by hydrogen bonds) are detached from their backbones. If the pair rotates before it is reinserted in the molecule, the resulting transver- sional mutation would be termed a rotational base substitution. Many radiation-induced point muta- tions may result in this way. rotation technique See photographic rotation tech- nique. Roundup the trade name of a glycophosphate her- bicide (produced by the Monsanto Company) that kills most plants. When sprayed on weeds, the glyco- phosphate is absorbed by the leaves, enters the cells, and inactivates a critical enzyme. Plants cannot syn- thesize certain essential amino acids without this en- zyme. The target enzyme does not occur in animals, so Roundup is not toxic to humans.

The herbicide is rapidly degraded in the soil. Monsanto next pro- duced crops that were genetically engineered to carry resistance genes for glycophosphate. Seeds for these crops are sold under the trade names Roundup Ready corn, Roundup Ready cotton, Roundup Ready potatoes, Roundup Ready soy beans, etc. See GMO, transgenic plants. rough endoplasmic reticulum See endoplasmic re- ticulum. Rous sarcoma virus (RSV) the first oncogenic vi- rus to be discovered. It is an RNA virus that induces tumors in chickens. The RSV was one of the first retroviruses shown to produce a reverse transcript- ase (q.v.).

The genes gag, pol, and env, which charac- terize all retroviruses, were first identified in this vi- rus. The RSV genome also contains src, an oncogene, so named because it induces sarcomas. The src gene codes for a protein kinase, pp60v-src (q.v.), which is localized in the plasmalemma. Vertebrate cells con- tain a gene homologous to the src gene. To distin- guish the two, the viral gene is abbreviated v-src and the cellular gene c-src. The two genes differ in that v-src has an uninterrupted coding sequence, whereas c-src contains seven exons separated by six introns. The c-src gene is a proto-oncogene (q.v.).

See Appen- dix C, 1910, Rous; 1970, Temin and Mizutani; 1975, Wang et al.; 1978, Collett and Erickson; 1981, Par- ker, Varmus, and Bishop; 1989, Bishop and Varmus; oncogene hypothesis, retroviruses. royal hemophilia classical hemophilia (q.v.) trans- mitted by a defective X chromosome first carried by Queen Victoria of Great Britain and passed on to plague three generations of European royalty. R17 phage a small RNA androphage (q.v.). See bacteriophage. rpo See RNA polymerase. R (resistance) plasmid an extrachromosomal DNA molecule that confers on bacteria resistance to one or more antibiotics. It consists of two compo- nents: the resistance transfer factor (RTF) required for transfer of the plasmid between bacteria, and the

r-determinants (genes conferring antibiotic resis- tance). R plasmids were first isolated from strains of Shigella dysenteriae that showed resistance to multi- ple antibiotics. See Appendix C, 1960, Watanabe and Fukusawa. rRNA ribosomal RNA. See ribosome. rRNA transcription unit See Miller trees. r strategy a type of life cycle exploiting high re- productive rate to achieve survival. See r and K selec- tion theory. RSV Rous sarcoma virus (q.v.). rtDNA See rDNA.

RTF resistance transfer factor. See R plasmid. rTU rRNA transcription unit. See Miller trees. RuBisCO an abbreviation for the enzyme ribulose- 1,5-bisphosphate carboxylase-oxygenase (q.v.). ruffled edges See lamellipodia. ruminant mammals even-toed hoofed mammals of a type that chews a cud. They include cattle, sheep, and goats and their relatives. runner a procumbent shoot that takes root, form- ing a new plant that eventually is freed from connec- tion with the parent by decay of the runner. The runner serves as a vegetative propagule. See Frageria, modular organisms. runt (run) a pair rule gene of Drosophila residing at 1-65. The run gene interacts with many other genes that regulate embryonic development (bicoid, fushi tarazu, and hunchback are examples).

Runt is expressed in subsets of neuroblasts in each neurom- ere at early stages of neurogenesis. The run gene en- codes a transcription factor containing 509 amino acids that is localized in the nucleus. This Runt pro- tein contains a DNA-binding motif 128 amino acids long. This Runt domain is also found in the protein encoded by lozenge (q.v.). Many other transcriptional regulators produced by both invertebrates and verte- brates contain Runt domains. In humans the symbol RUNX (RUNT-related) is given to these proteins, and one of them, RUNX1 (also called AML1) con- trols the differentiation of leucocytes. See acute my- eloid leukemia 1 gene, zygotic segmentation mutants. runting disease a pathological condition seen in young experimental animals inoculated with alloge- neic immunocompetent cells that produce a graft- versus-host reaction (q.v.). rut the period of sexual activity; estrus.

s 1. selection coefficient (q.v.) 2. standard devia- tion (q.v.) 3. sedimentation coefficient (q.v.) 4. second. S 1. Svedberg unit (q.v.) 2. Silurian. 3. sulfur. 4. DNA synthesis phase of the cell cycle (q.v.). 35S a beta-emitting radioactive isotope of sulfur with a half-life of 87.1 days; commonly used to label proteins via their sulfur-containing amino acids cyste- ine and methionine. 35S was used in the famous Her- shey-Chase experiment of 1952. See Appendix C. S1, S2, S3, etc.

the representation for continued selfing (self-fertilization) of plants. S1 designates the generation obtained by selfing the parent plant; S2, the generation obtained by selfing the S1 plant, etc. Saccharomyces cerevisiae the species of budding yeast used by brewers and bakers. Strains of brewer’s and baker’s yeast have special properties. Baker’s yeast can raise bread five times faster than brewer’s yeast, but baker’s yeast gives beer an undesirable yeasty flavor and settles out poorly. When nutrients are plentiful, wild type strains of S. cerevisiae prolif- erate as diploid cells. If starved, they go through meiosis to form haploid spores.

These can later ger- minate and proliferate as haploid cells, or they can fuse to reform diploids. The demonstration that yeast DNA has a high content of AT relative to GC disproved the tetranucleotide hypothesis (q.v.). S. cerevisiae was the first eukaryote to have its entire genome sequenced. It contains 12,068,000 base pairs, divided among 17 chromosomes. Its 5,885 ORFs make up about 70% of the genome. Only about 4% of the genes contain introns (q.v.). This is unusual, since 40% of the genes of Schizosaccharomyces pombe (q.v.) contain introns. In S. cerevisae there are 140 rRNA genes in a large tandem array on chromosome 12.

The 40 genes for snRNAs and the 275 genes for tRNAs are dispersed among all the chromosomes. S. cerevisiae is a favorite species for studying the genetic regulation of progression through the cell cycle. See Appendix A, Fungi, Ascomycota; Appendix C, 1949, Ephrussi et al.; 1965, Holley et al.; 1970, Khorana et al.; 1973, Hartwell et al.; 1974, Dujon et al.; 1979, Cameron et al.; 1980, Clark and Carbon; 1985, Boeke et al.; 1989, Kaback, Steensma, and DeJonge; 1992, Oliver et al.; 1996, Goffeau et al.; 1999, Galit- sky et al.; 2000, Rubin et al.; Appendix E, Individual Databases; bud, cassettes, centromere, Chargaff rule, genetic code, mitochondrial DNA, omnipotent sup- pressors, orphan, petites, Plasmodium, Ty elements, universal code theory, yeast artificial chromosomes (YACs), yeast two-hybrid system. S-adenosylmethionine See 5-methylcytosine. SAGE See serial analysis of gene expression (SAGE).

Saint-Hilaire hypothesis the proposal by Etienne Geoffroy Saint-Hilaire that arthropods and verte- brates have a common body plan. However, the plan in the two groups is inverted, since the nervous sys- tem of an insect is ventral, while that of a mammal is dorsal. Recent studies on genes that control the dorsoventral differentiation of embryonic cells sup- port the idea that the pattern for the body axis turned upside down during the early evolution of chordates.

See Appendix C, 1822, Saint-Hilaire; 1994, Arendt and Nu¨bler-Jung. salivary gland chromosomes polytene chromo- somes found in the interphase nuclei of the salivary gland cells in larval diptera. These chromosomes un- dergo complete somatic pairing; consequently, the mature salivary gland chromosome consists of two homologous polytene chromosomes fused side by side. See Appendix C, 1881, Balbiani; 1912, Ram- bousek; 1933, Painter; 1934, Bauer; 1935, Bridges; Drosophila salivary gland chromosomes. salivary gland squash preparation 1. a rapid method of preparing insect polytene chromosomes for microscopic investigation without sectioning them. The organ is simply squashed in a drop of stain placed between slide and coverslip. See aceto- orcein. 2. a method of preparing giant polytene chromosomes for localization of specific DNA se- quences via in situ hybridization. Larval salivary glands are quickly squashed in an appropriate fixa- tive between slide and coverslip, frozen, and the coverslip removed. The squashed specimens are then dehydrated and prepared for hybridization with labeled nucleic acid probes.

See in situ hybrid- ization. S allele See self-sterility genes. Salmo the genus containing various fish species of economic importance; especially S. salar, the Atlan-

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