APPENDIX C: CHRONOLOGY
M. Grunstein and D. S. Hogness develop the colony hybridization method for the isolation of cloned DNAs containing specific DNA segments or genes. A. T. C. Carpenter identifies recombination nodules in Drosophila melanogas- ter. She points out the correlation between the number of RNs and the number of meiotic exchanges. D. Pribnow determines the nucleotide sequences of two independent bacterio- phage T7 promoters, and compares these and other known promoter sequences to form a model for promoter structure and function. E. M. Southern describes a method for transferring DNA fragments from agar- ose gels to nitrocellulose filters. The filters are subsequently hybridized to ra- dioactive RNA and the hybrids detected by autoradiography. W. D. Benton and R. W. Davis describe a rapid and direct method for screen- ing plaques of recombinant lambda bacteriophages that involves transfer of phage DNA to a nitrocellulose filter and detection of specific DNA sequences by hybridization to complementary labeled nucleic acids. F. Sanger and A. R. Coulson develop the “plus and minus” method for deter- mining the nucleotide sequences in DNA by primed synthesis with DNA poly- merase. M. C. King and A. C. Wilson point out that 99% of the proteins that have been studied in humans and chimpanzees have identical amino acid sequences. They conclude that the biological differences between these two species must be largely the result of mutations that involve regulatory rather than structural genes. G. Morata and P. A. Lawrence show in Drosophila that the engrailed mutation allows cells of the posterior wing compartment to mix with those of the ante- rior compartment. Therefore, the normal allele of this gene functions to define the boundary conditions between the sister compartments of the developing wing. B. Mintz and K. Illmensee inject XY diploid cells from a malignant mouse teratocarcinoma into mouse blastocysts that then are transferred to foster mothers. Cells derived from the carcinoma appear in both somatic and germ cells of some F1 males. When these are mated, some F2 mice contain marker genes from the carcinoma. The experiments demonstrate that the nuclei of teratocarcinoma cells remain developmentally totipotent, even after hundreds of transplant generations during which they functioned in malignant cancers. S. L. McKenzie, S. Henikoff, and M. Meselson isolate mRNAs for heat-shock proteins and show that they hybridize to specific puff sites on the Drosophila polytene chromosomes. L. H. Wang and three colleagues locate within the RNA genome of the Rous sarcoma virus the segment responsible for its oncogenic activity. G. Blobel and B. Dobberstein put forth the signal hypothesis. R. Dulbecco, H. Temin, and D. Baltimore receive Nobel Prizes for their studies on oncogenic viruses. H. R. B. Pelham and R. J. Jackson describe a simple and efficient mRNA- dependent in vitro translation system using rabbit reticulocyte lysates. W. Fiers and 11 colleagues complete their analysis of MS2 RNA. This is the first virus to have its genome sequenced from beginning to end.
R. V. Dippell shows in Paramecium that kinetosomes contain RNA (not DNA) and that RNA (not DNA) synthesis accompanies kinetosome reproduction. N. Hozumi and S. Tonegawa demonstrate that the DNA segments coding for the variable and constant regions of an immunoglobulin chain are distant from one another in the chromosomes isolated from mouse embryos, but the seg- ments are adjacent in chromosomes isolated from mouse plasmacytomas. They conclude that somatic recombination during the differentiation of B lympho- cytes moves the constant and variable gene segments closer together. W. Y. Kan, M. S. Golbus, and A. M. Dozy are the first to use recombinant DNA technology in a clinical setting. They develop a prenatal test for alpha thalassemia utilizing molecular hybridization techniques. P. M. Nurse, P. Thuriaux, and K. Nasmyth elucidate the genetic control of the cell division cycle in Schizosaccharomyces pombe. Among the genes that control mitosis are those that encode cyclin-dependent kinases. M. F. Gellert and three colleagues discover DNA gyrase to be the enzyme that converts a relaxed, closed, circular DNA molecule into a negatively supercoiled form. W. Y. Chooi shows that ferritin-labeled antibodies raised against proteins (iso- lated from rat ribosomes) bind to the terminal knobs of fibers extending from Miller trees (isolated from the ovarian nurse cells of Drosophila). This observa- tion proves that Miller trees are rRNA transcription units and shows that at least some ribosomal proteins attach to a precursor rRNA molecule before its transcription is completed. B. G. Burrell, G. M. Air, and C. A. Hutchison report that phage phiX174 contains overlapping genes. Formal guidelines regulating research involving recombinant DNA are issued by the National Institutes of Health in the United States. H. Boyer and R. Swanson found Genentech, an event which marks the begin- ning of the biotech industrial revolution. A. Efstratiadis and three colleagues are the first to enzymatically generate eu- karyotic gene segments in vitro. They synthesize double-stranded DNA mole- cules that contain the sequences transcribed into the mRNAs for the alpha and beta chains of rabbit hemoglobin. J. T. Finch and A. Klug propose that the 300 A˚ threads seen in electron micro- graphs of fragmented chromatin are formed by the folding of DNA-nucleo- some filaments into solenoids. L. H. Miller and three colleagues conclude that the Duffy blood group antigens (Fya and Fyb) serve as receptors for the merozoites of Plasmodium vivax and that individuals of blood group Fy−/Fy− are resistant to P. vivax infections be- cause their red cells lack these receptors. A. Knoll and E. S. Barghoorn find microfossils which they interpret as undergo- ing cell division in rocks 3,400 million years old. This discovery pushes back the age of life on earth to the lower Archean eon. J. B. Corliss and R. D. Ballard aboard Alvin, a deep-diving minisubmarine, dis- cover communities of hyperthermophilic bacteria, tube worms, clams, and other organisms, living in the Galapagos rift. E. M. Ross and A. G. Gilman show that adenylcyclase is regulated by a protein that binds GTP. This G protein is purified three years later and shown to be a heterotrimer.
K. Itakura and six colleagues chemically synthesize a gene for human somato- statin and express it in E. coli. This leads to the commercial production of the first artificial human protein, somatostatin. S. M. Tilghman and eight colleagues clone the first protein-encoding gene (mouse beta-hemoglobin) using bacteriophage lambda as a vector. C. Jacq, J. R. Miller, and G. G. Brownlee describe the presence of “pseudo- genes” within the 5S DNA cluster of Xenopus laevis oocytes. J. C. Alwine, D. J. Kemp, and G. R. Stark prepare diazobenzyloxymethyl (DBM) paper and describe methods for transferring electrophoretically sepa- rated bands of RNA from an agarose gel to the DBM paper. Specific RNA bands are then detected by hybridization with radioactive DNA probes, fol- lowed by autoradiography. Since this method is the reverse of that described by Southern (1975) in that RNA rather than DNA is transferred to a solid support, it has come to be known as “northern blotting.” F. Sanger and eight colleagues report the complete nucleotide sequence for the DNA genome of bacteriophage phiX174. E. W. Silverton, M. A. Navia, and D. R. Davies determine the three-dimen- sional structure of the human immunoglobulin molecule. M. Leffak, R. Grainger, and H. Weintraub show that “old” histone octamers remain intact during DNA replication and that “new” octamers consist entirely of proteins synthesized immediately before replication. C. Woese and G. E. Fox conclude from their studies of the nucleotide se- quences of the 16S rRNAs of certain newly discovered microorganisms that they should be placed in a domain separate from the other bacteria (the Archaea). W. Gilbert induces bacteria to synthesize useful nonbacterial proteins (insulin and interferon). A. M. Maxam and W. Gilbert publish the “chemical method” of DNA se- quencing. R. J. Roberts and P. A. Sharp lead groups that discover split genes in adenovirus 2. R-loop mapping by L. Chow and S. Berget shows the position of intron loops. Intervening noncoding segments are then described for genes that encode animal proteins, namely, the rabbit beta-globin gene (A. Jeffreys and R. A. Flavell) and the chicken ovalbumin gene (R. Breathnach, J. L. Mandel, and P. Chambon). J. Weber, W. Jelinek, and J. E. Darnell report that alternative splicing of non- consecutive DNA segments in the adenovirus-2 genome can produce multiple mRNAs. J. F. Pardon and five colleagues use neutron contrast matching techniques to demonstrate that in nucleosomes the DNA segment that attaches to the his- tone octamer is on the outside of the particle. J. Sulston and H. R. Horvitz work out the postembryonic cell lineages for Caenorhabditis elegans. J. Collins and B. Holm develop cosmids for cloning large DNA fragments. F. Lee and C. Yanofsky explain the mechanism of attenuation that takes place in the tryptophan operon of E. coli. R. S. Yalow receives a Nobel Prize for developing the radioimmunoassay proce- dure.
R. M. Schwartz and M. O. Dayhoff compare sequence data for a variety of proteins and nucleic acids from an evolutionarily diverse assemblage of pro- karyotes, eukaryotes, mitochondria, and chloroplasts. Their computer-gener- ated evolutionary trees identify the times during evolution when protoeukaryo- tic organisms entered into symbiosis with mitochondria and chloroplasts (about 2 and 1 billion years ago, respectively). W. Gilbert coins the terms intron and exon. T. Maniatis and seven colleagues develop a procedure for gene isolation, which involves construction of cloned libraries of eukaryotic DNA and screening these libraries for individual sequences by hybridization to specific nucleic acid probes. M. S. Collett and R. L. Erickson report that the product of the src gene of the Rous sarcoma virus is a protein kinase. W. Bender, R. Spierer, and D. Hogness describe a method for sequencing genes they call chromosome walking. E. B. Lewis concludes that the component genes in the bithorax complex have related functions in Drosophila segmentation and that they evolved from a smaller number of ancestral genes by their duplication and subsequent special- ization. C. Coulondre and three colleagues show that sites in the DNA of E. coli identi- fied as mutational hot spots contain the modified pyrimidine, 5-methylcyto- sine. V. B. Reddy and eight colleagues publish the complete nucleotide sequence for simian virus 40 and correlate the sequence with the known genes and mRNAs of the virus. Y. W. Kan and A. M. Dozy demonstrate the value of using restriction-frag- ment-length polymorphisms as linked markers for the prenatal diagnosis of sickle-cell anemia. C. A. Hutchison and five colleagues demonstrate that it is possible to introduce specific mutations at specific sites in a DNA molecule. E. H. Blackburn and J. G. Gall demonstrate that telomeres from Tetrahymena pyriformis consist of short DNA sequences (one strand containing AACCCC, the other TTGGGG) repeated tandemly 30 to 70 times. R. T. Schimke and three colleagues show that cultured mouse cells exposed to methotrexate develop resistance by amplifying the genes that encode the en- zymes that serve as the target for the drug. W. Arber, H. O. Smith, and D. Nathans share the Nobel Prize in Physiology or Medicine for the development of techniques utilizing restriction endonucle- ases to study the organization of genetic systems. P. D. Mitchell receives the Nobel Prize in Chemistry for his contribution to the understanding of biological energy transfer through the formulation of the chemiosmotic theory. J. G. Sutcliffe determines the complete 4,362 nucleotide pair sequence of the plasmid cloning vector pBR322. J. C. Avise, R. A. Lansman, and R. O. Shade successfully use restriction endo- nucleases to measure mitochondrial DNA sequence relatedness in natural pop- ulations.
The National Institutes of Health relax guidelines on recombinant DNA to allow viral DNA to be studied. S. Perdix-Gillot reports giant nuclei that are over 1 million-ploid in the cells of the silk gland of Bombyx mori. B. G. Barrell, A. T. Bankier, and J. Drouin report that the genetic code of human mitochondria has some unique, nonuniversal features. E. F. Fritsch, R. M. Lawn, and T. Maniatis determine the chromosomal arrange- ment and structure of human globin genes utilizing recombinant DNA tech- nology. J. R. Cameron, E. Y. Loh, and R. W. Davis discover transposable elements in yeast. N. Wexler and a group of Venezuelan colleagues begin a study of natives who are living in three fishing villages on the shore of Lake Maracaibo. Huntington disease is prevalent in this population, and eventually an eight-generation pedi- gree is constructed that contains over 11,000 people. Analyses of DNA samples of this group lead to the localization and eventual sequencing of the HD gene by MacDonald and coworkers in 1993. D. V. Goeddel and nine colleagues construct a gene that encodes human growth hormone (HGH) using recombinant DNA technology. The synthesized gene is expressed in E. coli under the control of the lac promoter and a polypep- tide having the properties of HGH is synthesized. M. R. Lerner and J. A. Steitz report the discovery of small nuclear ribonucleo- proteins (snurps). L. Olsson and H. S. Kaplan produce the first human hybridomas that manufac- ture a pure antibody in laboratory culture. A. Hershko, A. Ciechanover, I. A. Rose, and three other colleagues demon- strate that proteins destined to be destroyed undergo ATP-dependent conjuga- tion to a specific protein (APF-1). They later show that APF-1 is ubiquitin and that it delivers doomed proteins to the 26S proteasome where they are broken down into short peptides for reuse. The United States Supreme Court rules that genetically modified microorgan- isms can be patented. General Electric, on behalf of A. Chakrabarty, obtains a patent for a genetically engineered microorganism capable of consuming oil slicks. D. Lowe describes stromatolites from the Archean of Western Australia. They contain 3.8 billion-year-old fossils that resemble cyanobacteria. J. W. Gordon and four colleagues produce the first transgenic mice by direct injection of cloned DNA into the pronucleus of a fertilized egg. M. R. Capecchi describes a technique for efficient transformation of cultured mammalian cells by direct microinjection of DNA into cells with glass micropi- pettes. C. Woese and 10 colleagues publish the secondary structure for 16S ribosomal RNA. D. Botstein and three colleagues describe the method of using restriction frag- ment length polymorphisms to construct genetic linkage maps of the human genome.
W. F. Doolittle and C. Sapienza and, independently, L. E. Orgel and F. H. C. Crick point out that the genomes of all species are littered with DNA segments that contribute nothing to the fitness of the species and persist only because they are efficient replicators. These authors name this collection of DNA seg- ments selfish DNA and suggest that these DNAs represent the ultimate para- sites. H. Gronemeyer and O. Pongs demonstrate that, in Drosophila melanogaster sali- vary glands, beta ecdysone binds directly to sites on polytene chromosomes where ecdysone-inducible puffs occur. C. Nu¨sslein-Volhard and E. Wieschaus describe the isolation and characteriza- tion of zygotic segmentation mutations of Drosophila melanogaster. L. Clark and J. A. Carbon clone the gene that corresponds to the centromere of yeast chromosome 3. A. R. Templeton provides a new theoretical framework for speciation by the founder effect. Nobel Prizes in Physiology and Medicine go to G. D. Snell, J. Dausset, and B. Benacerraf for their contributions to immunogenetics. P. Berg, W. Gilbert, and F. Sanger receive Nobel Prizes in Chemistry for their contributions to the experimental manipulation of DNA. R. C. Parker, H. E. Varmus, and J. M. Bishop demonstrate that the tumorigenic properties of the Rous sarcoma virus are due to a protein encoded by the v-src gene. Cells from various vertebrates contain a homologous 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. L. Margulis publishes Symbiosis in Cell Evolution. Here she summarizes the evidence for the theory that organelles such as mitochondria, chloroplasts, and kinetosomes evolved from prokaryotes that lived as endosymbionts in the an- cestors of modern-day eukaryotes. R. Lande proposes a new model of speciation based on sexual selection on polygenic traits. This model results in a revival of interest in sexual selection. J. D. Kemp and T. H. Hall transfer the gene of a major seed storage protein (phaseolin) from beans to the sunflower via a plasmid of the crown gall bacte- rium Agrobacterium tumefaciens, creating a “sunbean.” T. R. Cech, A. J. Zaug, and P. J. Grabowski report the discovery of a self- splicing rRNA in Tetrahymena thermophila. This is the first demonstration that a macromolecule other than a protein can act as a biological catalyst. W. F. Anderson and three colleagues determine the three-dimensional struc- ture of the cro repressor at 2.8 A˚ resolution. G. Hombrecher, N. J. Brewin, and A. W. B. Johnson demonstrate that the ability of Rhizobium bacteria to nodulate legumes and fix atmospheric nitrogen is due to plasmid-linked genes. P. R. Langer, A. A. Waldrop, and D. C. Ward develop a procedure for synthe- sizing biotinylated DNA probes that hybridize normally with complementary DNA, providing an anchor for streptavidin-linked, color-generating systems. S. Anderson and 13 colleagues work out the complete nucleotide sequence and genetic organization of the human mitochondrial genome.
H. Sakano and three colleagues discover two segments in the heavy chain im- munoglobulin gene of the mouse, which serve as recognition sites for a somatic DNA recombinase. M. E. Harper and G. F. Saunders demonstrate that single-copy genes can be mapped on human mitotic chromosomes utilizing an improved in situ hybrid- ization technique. J. Banerji, S. Rusconi, and S. Schaffner show that the transcription of the beta- globin gene is enhanced hundreds of times when this gene is linked with certain SV40 nucleotide sequences that they name “enhancer sequences.” J. G. Gall and four colleagues localize histone mRNAs that are being tran- scribed on the lampbrush chromosomes of salamander oocytes. M. Chalfie and J. Sulston identify among the touch-insensitive mutants of Caenorhabditis elegans five genes that affect a specific set of six sensory neu- rons. K. E. Steinbeck and three colleagues demonstrate that the resistance of a weed, Amaranthus hybridus, to triazine herbicides is controlled by a chloroplast gene that encodes a polypeptide to which the herbicide binds. Resistant strains of the weed produce a modified gene product that fails to bind triazine. J. D. Walker sequences the eight genes of the E. coli atp operon. These encode the protein subunits of ATP synthase. Eli Lilly and Company markets a Genentech-licensed, recombinant, human in- sulin. This is the first product generated by this new technology. E. P. Reddy and three colleagues report that the genetic change that leads to the activation of an oncogene carried by a line of human bladder carcinoma cells is due to a single base substitution in this gene. The result is the incorpora- tion of valine instead of lysine in the 12th amino acid of the protein encoded by the oncogene. P. Goelet and five colleagues determine the complete nucleotide sequence for the RNA genome of the tobacco mosaic virus. P. M. Bingham, M. G. Kidwell, and G. M. Rubin show that P strains of Dro- sophila contain 30 to 50 copies per genome of a transposable P element. This is the cause of hybrid dysgenesis. Then A. C. Spradling and Rubin demonstrate that cloned P elements, when microinjected into Drosophila embryos, become integrated into germ-line chromosomes and that P elements can be used as vectors to carry DNA fragments of interest into the Drosophila germ line. E. R. Kandel and J. G. Schwartz utilize the gill-withdrawal reflex in Aplysia to study the molecular control of memory formation. They eventually show that the long-term facilitation of sensory neurons requires the activation of cAMP- responsive memory genes. S. B. Prusiner shows that the infectious agent that causes scrapie is a protein, which he calls a prion. A. Klug receives the Nobel Prize for his contributions to the analysis of crystal- line structures of biological importance, especially virus particles, tRNA, and nucleosomes. E. A. Miele, D. R. Mills, and F. R. Kramer construct the first recombinant RNA molecule by inserting a synthetic foreign deca-adenylic acid into a variant of the RNA genome of phage Q beta via the action of the Q beta replicase.
H. J. Jacobs and six colleagues report the presence of promiscuous DNA in the sea urchin. T. Hunt and four colleagues demonstrate that sea urchin eggs contain a mater- nal mRNA which encodes a protein that is synthesized after fertilization and is cyclically destroyed and resynthesized during cleavage divisions. They name the protein cyclin. I. S. Greenwald, P. W. Sternberg, and H. R. Horvitz demonstrate that the lin- 12 mutant of Caenorhabditis functions as a developmental control gene. M. Oren and A. J. Levine isolate and identify a cDNA of the p53 mRNA from a SV-40-transformed mouse cell line. S. D. Gillies and three colleagues show that a tissue-specific enhancer is located in the first intron of the heavy-chain immunoglobulin gene. W. Bender and seven colleagues sequence genes in the bithorax complex of Dro- sophila and show that spontaneous mutations by bx, Ubx, and bxd are associ- ated with insertions of transposable elements. M. P. Scott and six colleagues sequence another group of segment identity genes and work out the organization of the Antennapedia locus. G. N. Godson and four colleagues clone the gene that encodes the circumspo- rozoite protein of Plasmodium knowlesi, and they show that the protein con- tains a repetitive epitope that serves as a decoy to the host immune system. C. Guerrier-Takada and four colleagues show that ribonuclease P consists of one protein and one RNA subunit and that the latter is the catalytic subunit. L. Montagnier in France and R. Gallo in the United States lead teams that independently publish accounts of the discovery of the virus that causes AIDS. M. Kimura and T. Ohta estimate 1.8 × 109 years as the time of divergence of eukaryotes and prokaryotes through comparative studies of the nucleotide sequences of 5S rRNAs from humans, yeasts, and bacteria. M. Rassoulzadegan and six colleagues isolate a recombinant DNA clone from the polyoma virus that immortalizes cultured fibroblast cells from rat embryos. They also show that only the amino-terminal portion of the protein encoded by the viral gene carries the immortalizing function. R. F. Doolittle and six colleagues demonstrate that the simian sarcoma virus oncogene, v-sis, is derived from the gene encoding a platelet-derived growth factor. E. Hafen, M. Levine, and W. J. Gehring work out a technique for in situ hy- bridization of labeled DNA probes to RNA transcripts in frozen tissue sections. They succeed in localizing transcripts of homeotic genes to specific regions of developing Drosophila embryos. R. Mann, R. C. Mulligan, and D. Baltimore genetically engineer the Moloney murine leukemia virus so that it can be used safely as a vector in gene transfer experiments with mammalian hosts. B. McClintock receives the Nobel Prize for her discovery of transposable ge- netic elements. D. C. Schwartz and C. R. Cantor show that pulsed field gradient electrophore- sis can be used to separate DNA fragments as large as 2,000 kbp. This method overcomes the limitation of agarose gel electrophoresis, which can only sepa- rate molecules of much smaller sizes (50 kbp or less).