Barbara McClintock, 1902-1992.
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Genetics↗
Biomedical subjects
J A Shapiro
Publications and source records attributed to J A Shapiro.
Natural genetic engineering in evolution.
The results of molecular genetics have frequently been difficult to explain by conventional evolutionary theory. New findings about the genetic conservation of protein structure and function across very broad taxonomic boundaries, the mosaic structure of genomes and genetic loci, and the molecular mechanisms of genetic change all point to a view of evolution as involving the rearrangement of basic genetic motifs. A more detailed examination of how living cells restructure their genomes reveals a wide variety of sophisticated biochemical systems responsive to elaborate regulatory networks. In some cases, we know that cells are able to accomplish extensive genome reorganization within one or a few cell generations. The emergence of bacterial antibiotic resistance is a contemporary example of evolutionary change; molecular analysis of this phenomenon has shown that it occurs by the addition rearrangement of resistance determinants and genetic mobility systems rather than by gradual modification of pre-existing cellular genomes. In addition, bacteria and other organisms have intricate repair systems to prevent genetic change by sporadic physicochemical damage or errors of the replication machinery. In their ensemble, these results show that living cells have (and use) the biochemical apparatus to evolve by a genetic engineering process. Future research will reveal how well the regulatory systems integrate genomic change into basic life processes during evolution.
Animals↗
Differential action and differential expression of DNA polymerase I during Escherichia coli colony development.
A mini-Tn10 insertion in the polA cistron (polA2099) was isolated in a search for mutations that affect patterned Mudlac replication in colonies. The polA2099 mutation had a dramatic effect on cell morphogenesis during the first few hours of microcolony development. Abnormal microcolonies containing filamentous cells were produced as a result of SOS induction. Despite gross abnormalities in early microcolonies, mature polA2099 colonies after 2 to 4 days were morphologically indistinguishable from Pol+ colonies, and 44-h polA2099 colonies displayed a cell size distribution very similar to that of Pol+ colonies. These results suggested the involvement of a protective factor produced during colony growth that compensated for the polA deficiency. The action of a diffusible substance that stimulates growth of polA2099 microcolonies was shown by spotting dilute polA2099 cultures next to established colonies. Differential transcription of polA during colony development was visualized by growing colonies containing polA-lacZ fusions on beta-galactosidase indicator agar. When polA-lacZ colonies were inoculated next to established colonies, a diffusible factor was seen to inhibit polA transcription during the earliest stages of colony development. These results show that a basic housekeeping function, DNA polymerase I, is subject to multicellular control by the changing conditions which the bacteria create as they proliferate on agar.
Bacteriophage lambda↗
Cloning and expression of IDDM-specific human autoantigens.
A DNA cloning approach was taken to identify islet cell protein antigens that are recognized specifically by insulin-dependent diabetes mellitus (IDDM) sera. A human islet cDNA library was generated and screened with diabetic sera. In this article, identification of two clones is described. Proteins expressed by these lambda phages appeared to react specifically with newly diagnosed diabetic sera. Islet cell antibody 12 (ICA12) was tested by Western blotting. ICA512 was not reactive with sera in the Western format but was specifically immunoprecipitated by diabetic sera from an Escherichia coli extract.
Autoantigens↗
Pattern and control in bacterial colony development.
As we learn more about bacterial life in the laboratory and in nature, we increasingly appreciate that they are highly sensitive and sophisticated organisms. One of the principal new insights has been the appreciation that bacteria are interactive and form organized, differentiated multicellular communities. Colonies produced on laboratory media by the standard research bacterium, Escherichia coli, are excellent examples. The organization of these colonies can be visualized in the microscope, by macrophotography, and by the use of special dyes and genetic engineering techniques to reveal patterns of differential gene expression. Observation of the dynamics of colony growth, and the response of colonies to experimental disruptions of normal development, indicate that control systems work to produce the regular patterns observed. The effects of obstacles and of other colonies on gene expression patterns indicate that non-linear responses to chemical gradients in the substrate play an important coordinating role in colony development.
Bacteria↗
Genomes as smart systems.
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Gene Expression↗
Action of a transposable element in coding sequence fusions.
The original Casadaban technique for isolating fused cistrons encoding hybrid beta-galactosidase proteins used a Mucts62 prophage to align the upstream coding sequence and lacZ prior to selection. Kinetic analysis of araB-lacZ fusion colony emergence indicated that the required DNA rearrangements were regulated and responsive to conditions on selection plates. This has been cited as an example of "directed mutation." Here we show genetically that the MuA and integration host factor (IHF) transposition functions are involved in the formation of hybrid araB-lacZ cistrons and propose a molecular model for how fusions can form from the initial strand-transfer complex. These results confirm earlier indications of direct Mu involvement in the fusion process. The proposed model explains how rearranged Mu sequences come to be found as interdomain linkers in certain hybrid cistrons and indicates that the fusion process involves a spatially and temporally coordinated sequence of biochemical reactions.
Base Sequence↗
General practitioners' attitudes towards AIDS and their perceived information needs.
A short questionnaire on general practitioners' self perceived and actual knowledge of AIDS and their attitudes to the illness was sent to 1824 general practitioners throughout the United Kingdom. The rate of response was 70%. Women doctors, those who trained overseas, and those who were married tended to have less positive attitudes towards patients with HIV and AIDS, whereas younger doctors, trainers, and members of the Royal College of General Practitioners were more understanding, better informed, and had more positive attitudes. Doctors with the least knowledge about HIV and AIDS and the most negative attitudes towards the illness would benefit from further education, which would be most effectively delivered through the professional journals, the Department of Health and the charitable AIDS organisations.
Acquired Immunodeficiency Syndrome↗
Rapid identification of microorganisms by nucleic acid hybridization after labeling the test sample.
A novel method for rapidly identifying microorganisms has been developed. This method employs a monoadduct-forming furocoumarin derivative, which can photochemically label nucleic acids. The labeled nucleic acid can, in turn, be hybridized simultaneously to a panel of immobilized probe DNAs arrayed as dots on a solid support such as nitrocellulose. This procedure offers several advantages over more conventional hybridization techniques in that sample nucleic acids can be photolabeled without substantial sample preparation and that identification can be achieved by a single, rapid hybridization reaction.
Bacterial Typing Techniques↗
Escherichia coli K-12 cell-cell interactions seen by time-lapse video.
The high degree of organization in mature bacterial colonies suggests specific interactions between the cells during colony development. We have used time-lapse video microscopy to find evidence for cell-cell interactions. In its initial stages, Escherichia coli K-12 colony morphogenesis displayed control of the geometry of cell growth and involved intimate side-by-side associations. When microcolonies developed from isolated single bacteria, a directed process of elongation and division resulted in the appearance of a symmetrical four-cell array. When growth began with separate but nearby bacteria, the daughters of different cells elongated towards each other and also lined up side by side. Interactions between microcolonies containing several hundred or more bacteria were visible several hours later. Control of cell morphogenesis at later stages of microcolony development was strain specific. These results show that E. coli K-12 cells respond to each other and adjust their cellular morphogenesis to form multicellular groups as they proliferate on agar.
Cell Division↗
Differential activity of a transposable element in Escherichia coli colonies.
In Escherichia coli colonies, patterns of differential gene expression can be visualized by the use of Mu d(lac) fusion elements. Here we report that patterned beta-galactosidase expression in colonies of strain MS1534 resulted from a novel mechanism, spatially localized replication of the Mu dII1681 element causing lacZ transposition to active expression sites. Mu dII1681 replication did not occur constitutively with a fixed probability but was dependent on the growth history of the bacterial population. The bacteria in which Mu dII1681 replication and lacZ transposition had occurred could no longer form colonies. These results lead to several interesting conclusions about cellular differentiation during colony development and the influence of bacterial growth history on gene expression and genetic change.
Blotting, Southern↗
Expression of a synthetic gene encoding human insulin-like growth factor I in cultured mouse fibroblasts.
A synthetic gene encoding human insulin-like growth factor I (hIGF-I) was assembled and inserted into an expression vector containing the cytomegalovirus immediate early (CMV-IE) transcriptional regulatory region and portions of the bovine growth hormone gene. The recombinant plasmid encodes a 97 amino acid fusion protein containing the first 27 amino acids of the bovine growth hormone precursor and the 70 amino acids of hIGF-I. This plasmid, when transiently introduced into cultured mouse fibroblasts, directs synthesis of the fusion protein, subsequent proteolytic removal of the bovine growth hormone signal peptide, and secretion of hIGF-I into the culture medium. Conditioned medium from transfected cells inhibits binding of 125I-labeled IGF-I to type I IGF receptors on human placental membranes and to acid-stable human serum carrier proteins. The recombinant hIGF-I produced is biologically active, as monitored by the stimulation of DNA synthesis in vascular smooth muscle cells.
Amino Acid Sequence↗
Mechanisms of DNA reorganization in bacteria.
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Allosteric Regulation↗
Molecular model for the transposition and replication of bacteriophage Mu and other transposable elements.
A series of molecular events will explain how genetic elements can transpose from one DNA site to another, generate a short oligonucleotide duplication at both ends of the new insertion site, and replicate in the transposition process. These events include the formation of recombinant molecules which have been postulated to be intermediates in the transposition process. The model explains how the replication of bacteriophage Mu is obligatorily associated with movement to new genetic sites. It postulates that all transposable elements replicate in the transposition process so that they remain at their original site while moving to new sites. According to this model, the mechanism of transposition is very different from the insertion and excision of bacteriophage lambda.
Coliphages↗
Integration and excision of prophage lambda mediated by the IS 1 element.
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Bacteriophage lambda↗
Transposition of Tn7 in Pseudomonas aeruginosa and isolation of alk::Tn7 mutations.
Conjugal crosses with Pseudomonas aeruginosa donors carrying the CAM-OCT and RP4::Tn7 plasmids result in transfer of the Tn7 trimethoprim resistance (Tp(r)) determinant independently of RP4 markers. All Tp(r) exconjugants which lack RP4 markers have CAM-OCT genes and therefore must have received CAM-OCT::Tn7 plasmids formed by transposition of Tn7 from RP4::Tn7 to CAM-OCT. Most crosses yield exconjugants carrying mutant CAM-OCT plasmids which no longer determine either camphor or alkane utilization and thus appear to carry Tn7 inserts in the cam or alk loci, respectively. Transduction and reversion experiments indicated that at least 13 alkane-negative, camphor-positive, Tp(r) CAM-OCT::Tn7 plasmids carry an alk::Tn7 mutation. Determination of linkage between the alk mutation and the Tp(r) determinant of Tn7 on these plasmids is complicated by the presence of multiple copies of the Tn7 element in the genome. Generalized transduction will remove Tn7 from a CAM-OCT alk::Tn7 plasmid to yield alk(+) cells which carry no Tp(r) determinant on the CAM-OCT plasmid (as shown by transfer of the plasmid to a second strain). But the transduction to alk(+) does not remove all Tp(r) determinants from the genome of the recipient cell because the alkane-positive transductants remain trimethoprim resistant. Thus, it appears that copies of Tn7 can accumulate in the genome of P. aeruginosa (CAM-OCT alk::Tn7) strains without leaving their original site. This result is consistent with transposition models that involve replication of the transposable element without excision from the original site.
Alkanes↗
Chromosomal integration of phage lambda by means of a DNA insertion element.
Phage lambdacam112, which contains the chloramphenicol resistance transposon Tn9 and has a deletion of attP and the int gene, will lysogenize Escherichia coli K-12. Prophage integration occurs at different chromosomal sites, including lacY and malB, but not at attB. All lambdacam112 prophages are excised from the chromosome after induction but with various efficiencies for different locations. Heteroduplex analysis of lambdaplacZ transducing phages isolated from a lacY::lambdacam112 prophage reveals an insertion sequence 1 (IS1) element at the joint of viral and chromosomal DNA. Two lines of evidence indicate that lambdacam112 encodes an excision activity that recognizes the IS1 element: (i) prophage derepression increases the frequency of excision from lacY to yield lac+ revertants, and (ii) lambdacam112 infection increases reversion of a galT::IS1 mutation about 50-fold. Our results indicate that the IS1 termini of TN9 can replace attP as a site for lambda insertion in the bacterial chromosome and that excision events are catalyzed by an IS1-encoded protein under lambda repressor and N gene control.
Base Sequence↗
Sequence analysis of oligodeoxyribonucleotides by mass spectrometry. 1. Dinucleoside monophosphates.
The base components of underivatized oligodeoxynucleotides can be determined qualitatively by mass-spectral analysis at the nanogram level. The thermal and electron-impact conditions of the spectrometer allow the cleavage of the phosphodiester bonds of the oligonucleotide chain, resulting in fragments (I), whose mass identifies the base, and other fragments (II), which contain the purine or pyrimidine base plus portions of the deoxyribose and the phosphate moieties. A study of 16 dinucleoside monophosphates indicates that the relative intensities of the m/e values of the type II fragments are significantly and reproducibly different for sequence isomers. From the complex spectra of dinucleoside monophosphates, specific ions for each mono-nucleotide residue have been selected which reveal the location (5' or 3' terminus) of the bases in the dinucleoside monophosphate. These ions appear in spectra of deoxyribonucleic acid fragments as well as in model compounds. A simple computer program has been devised which utilizes ion ratio values to determine sequence. The method is applicable to oligonucleotides of longer length.
Base Sequence↗