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- Volume 29, Issue 5_Part_2, September 1980
The American Journal of Tropical Medicine and Hygiene - Volume 29, Issue 5_Part_2, September 1980
Volume 29, Issue 5_Part_2, September 1980
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Introductory Remarks
Pages: 1023–1024More LessI welcome you to this Workshop on the Application of the Recombinant DNA Technology to Protozoology sponsored by the National Institute of Allergy and Infectious Diseases. The subject of my talk is based on the title of the branch of this Institute in which I am located—Molecular Microbiology and Parasitology.
Our organization, the National Institute of Allergy and Infectious Diseases, traces its origin back to 1887 when the Laboratory of Hygiene was established at the Marine Hospital on Staten Island, New York. In 1891, the Laboratory of Hygiene was redesignated the Hygienic Laboratory and moved to Washington, D.C. In 1930, the Hygienic Laboratory became the National Institute of Health which moved to its present location in Bethesda, Maryland, in 1938. Ten years later this, in turn, became the National Microbiology Institute which, since 1955, has been the National Institute of Allergy and Infectious Diseases.
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An Introduction to Antigenic Variation in Trypanosomes
Pages: 1027–1032More LessAbstractThe salivarian trypanosomes have a unique capacity for antigenic variation at the cell surface. This phenomenon is their primary mechanism for evasion of the host's immune response. Variation is mediated through alternate expression of an extensive repertoire of variant surface glycoproteins (VSGs). Extensive amino acid sequence diversity is responsible for the antigenic diversity of VSGs. All the isolated VSGs of Trypanosoma brucei studied also contain an immunologically cross-reacting glycosyl side chain at the C-terminus, which probably represents a recognition site for proteolytic processing of the hydrophobic putative membrane-binding tail present on the synthesized molecule but not so far found on purified VSGs.
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The Genes for Variant Antigens in Trypanosomes
Pages: 1033–1036More LessAbstractWe have studied the mechanism of antigenic variation by using DNA complementary to the messenger RNAs for four variant surface glycoproteins of Trypansoma brucei. Pure complementary DNAs were obtained by cloning as recombinant DNA in Escherichia coli. Using these complementary DNAs as hybridization probes, we have analyzed the genes for these variant surface glycoproteins. The results provide new information on the origin and evolution of antigenic variation, and on the mechanism involved in switching from one antigenic type to another.
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Analyses of Variable Antigen Gene Rearrangements in Trypanosoma Brucei *
Pages: 1037–1042More LessAbstractFor the purpose of investigating the genetic basis of antigenic variation in Trypanosoma brucei, we have analyzed the structure of the genome surrounding the gene coding for one T. brucei variable antigen (IL Tat 1.2) in several T. brucei clones expressing this and other variable antigens. In each case there are two copies of the gene. We found no evidence of an extra copy associated with the expression of this gene. Differences were found between the two copies in a single clone, and between the copies in different clones. The differences could be explained by insertion and deletion of various lengths of DNA in a region beyond the C-terminal end of the gene. Differences in genomic structure were found even between clones expressing the same antigen, whether IL Tat 1.2 or another. Thus, no feature of the rearrangements observed can be correlated with the expression of a particular antigen.
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Structural Analysis of Variant and Invariant Genes in Trypanosomes
Pages: 1043–1049More LessAbstractVariant surface antigens have been purified from variants of a newly developed serodeme of Trypanosoma brucei 164, and found to be distinct by immunological and molecular criteria. cDNA clones containing variant and invariant gene sequences were isolated by using RNA from different variants and cloned DNAs from other systems as probes. These trypanosome and heterologous cloned DNAs were used as probes of trypanosome gene structure and activity. Regulation of variant antigen gene expression is at the level of transcription, and the variant antigen genomic sequences differ in their arrangement between the variants. Chick α- and β-tubulin sequences hybridize to trypanosome-tubulin gene sequences, while chick β-actin sequences do not. A 1650 bp β-tubulin trypanosome cDNA has been isolated. The trypanosome α- and β-tubulin sequences appear to be clustered in the genome. Sequence rearrangement of tubulin genes does not occur during antigenic variation.
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The Kinetoplast DNA of the Hemoflagellate Protozoa
Pages: 1053–1063More LessAbstractThe kinetoplast DNA of Leishmania tarentolae and Trypanosoma brucei was studied in terms of genetic organization and transcriptional activity. Several minor sequence classes of minicircles from L. tarentolae were cloned in a bacterial plasmid and were compared in terms of sequence organization. The cloned minicircles were characterized by the possession of a constant region of at least 91 nucleotides and a variable region. Minicircles from Trypanosoma brucei strain 366D were also cloned. Fragments of the maxicircle DNA from both species were also cloned in pBR322. No homology with the cloned minicircles was apparent. Several maxicircle transcripts, in addition to the 9 and 12s presumptive ribosomal RNAs, were observed in L. tarentolae. The 9 and 12s RNA genes were also mapped on the T. brucei maxicircle. Sequence homology between the L. tarentolae 9 and 12s RNA genes and the T. brucei 9 and 12s RNA genes was observed. A culture system was developed to study the developmental change of cultured bloodstream forms of T. brucei into procyclic forms. This developmental system is amenable for the study of the role of the kinetoplast DNA in the extensive mitochondrial biogenesis that occurs at this time.
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The Replication of Kinetoplast DNA
Pages: 1064–1069More LessAbstractKinetoplast DNA is the mitochondrial DNA of trypanosomatids. This DNA consists primarily of thousands of small minicircles which are linked together to form a giant network. Replication of this DNA involves release of individual minicircles from the network to form free minicircles. The free minicircles then replicate and the two progeny are reattached to the network. When all minicircles within the network have replicated, the double-sized network divides to form two progeny structures which are each identical to the parent network.
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Characterization of Pathogenic Trypanosomatidae by Restriction Endonuclease Fingerprinting of Kinetoplast DNA Minicircles
Pages: 1070–1074More LessAbstractA simple protocol was developed for the routine preparation of a kinetoplast DNA fraction from trypanosomatids. The digestion of this DNA with selected restriction endonucleases, followed by the electrophoretic analysis of the fragments on polyacrylamide gradient gels, yielded characteristic patterns that could be used for the intrinsic characterization of stocks (populations derived by serial passage in vivo and/or in vitro from a primary isolation, without any implication of homogeneity or characterization), strains (sets of populations originating from a group of trypanosomes of a given species or subspecies present at a given time in a given host or culture, and defined by the possession of one or more designated characters), and clones (trypanosomes derived from a single individual by binary fission) of certain pathogenic hemoflagellates.
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Kinetoplast DNA of Normal and Mutant Trypanosoma Brucei
Pages: 1075–1081More LessAbstractKinetoplast DNA (kDNA) sequence organization, transcription, and alterations in dyskinetoplastic (Dk) mutants were examined in Trypanosoma brucei, using physically isolated and recombinant kDNA sequences. Maxicircles renatured as a single sequence class and had no homology with minicircles. Total minicircle complexity was greater than 300 kb. Minicircle sequence organization is complex. Different minicircles have sequences in common and comprise varying fractions of the kDNA network. Transcripts of the same maxicircle restriction fragments, representing most of the maxicircle, were detected in both bloodstream and cultured procyclic form RNA. Presumptive mitochondrial ribosomal RNA coding sequences were localized to a specific maxicircle segment. No minicircle transcription was detected. All Dk mutants examined had substantial reduction of kDNA sequences. No kDNA sequences could be detected in one mutant examined in detail. Another Dk mutant was found to contain sequences homologous to kDNA. These DNA sequences had the same electrophoretic mobility as maxicircle and minicircle restriction fragments.
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New Insights into How Bacteria Take up DNA during Transformation
Pages: 1085–1088More LessAbstractHaemophilus influenzae possesses an efficient natural transformation system. Under appropriate growth conditions, every cell in a culture acquires the competence to take up several molecules of DNA. Only donor DNAs from Haemophilus species are taken up; foreign DNAs are excluded. Specific recognition is achieved through the interaction of a receptor protein present on the recipient cell membrane with an 11 base pair sequence present at high frequency in the donor DNA.
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A New Selective Agent for Eukaryotic Cloning Vectors
Pages: 1089–1092More LessAbstractAn aminoglycoside antibiotic, G418, has been shown to be an inhibitor of many pro- and eukaryotes at concentrations from 1–300 µg/ml. A bacterial R-plasmid determinant that phosphorylates and inactivates antibiotic G418 can be introduced into yeast by transformation and expresses resistance to G418. It is suggested that this combination of antibiotic and dominant resistance mechanism may be useful in recombinant DNA studies as a cloning selection in eukaryotes.
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Eukaryotic Gene Transfer and the New Genetics
Pages: 1093–1095More LessAbstractThe function of specific eukaryotic DNA sequences can be assessed by the following procedure. First, the gene of interest is purified, usually by cloning in bacteria, and its structure is determined. Second, biochemical techniques are used to introduce mutations into the regions of interest. Finally, the normal and mutant genes are introduced into an appropriate eukaryotic cell where their expression can be examined in detail. In this paper I review some of the techniques that can be used for gene transfer, and speculate on how they might be applied to parasites.
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Site-Specific Recombination in Bacteriophage λ: Structural Analyses of Reactive DNA Sequences
Pages: 1099–1106More LessAbstractSite-specific integrative recombination in bacteriophage λ involves unequal partners. The minimal phage att site is composed of approximately 240 base pairs and has four distinct Int binding sites that differ in size and response to heparin challenge. There appear to be two size classes of Int binding sites, approximately 30–35 base pairs and 15 base pairs. The sites at the common core and in the P′ arm are of the former class. Two sites in the P arm are of the latter class. Thus far, three of the four sites have been shown to be necessary for att site function. In contrast, the minimal sequence required for a phage att site partner (such as the bacterial att site) may not be much larger than the 15 base pair common core. We have suggested a model in which integrative recombination involves two unequal partners; accordingly the phage att site is referred to as the “donor” and the bacterial att site, or its analogue, is referred to as the “recipient.”
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Identification of Ecotropic Proviral Sequences in Twelve Inbred Mouse Strains
Pages: 1107–1110More LessAbstractA specific probe for detecting ecotropic murine leukemia virus sequences was constructed by cloning a 400 base-pair DNA, corresponding to a portion of the env region of the AKR ecotropic virus. We have employed this probe to detect the presence or absence of ecotropic retroviral DNA sequences in 12 inbred strains of mice using the Southern blotting hybridization procedure. Our results show that between 2 and 6, and 1–2 copies of the ecotropic viral genome are present in mice with high or low incidence of leukemia, respectively. Ecotropic viral sequences are either absent or present in partial copies in mouse strains which heretofore failed to yield ecotropic viruses.
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Recombinant DNA Studies on the Structure, Evolution, and Developmental Expression of Structural Gene Families
Pages: 1111–1116More LessAbstractThe structure of silkmoth chorion genes and their organization within the chromosomes are summarized. The multiple chorion genes are members of homologous gene families, and are physically linked. Their detailed structure and organization suggest mechanisms for their evolution and for the regulation of their expression.
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Determination of the Identity and Organization of Messenger RNAs Within DNA
Pages: 1117–1122More LessAbstractWe outline methods for identifying DNAs containing sequences complementary to specific mRNAs, and also provide a number of complementary approaches for mapping the arrangement of mRNAs along the DNA. These methods, together with S1 nuclease mapping and the direct visualization of R-loops with the electron microscope, provide a comprehensive approach to defining the architecture of mRNAs coding for specific polypeptides and the arrangement of RNA transcripts along the genome. This detailed cartographic information can then be used to study the steps in the processing of mature mRNAs and determine the modes by which the expression of specific genes is regulated.
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Prospects and Problems in the Use of Recombinant DNA for the Production of a Malaria Vaccine *
Pages: 1125–1128More LessAbstractRecent advances in malaria immunology and the discovery of an in vitro culture method for Plasmodium falciparum have opened the way for the feasibility of formulating a vaccine as a control measure for this most virulent species of human malaria. The successful use of recombinant DNA technology for the production of scarce cellular constituents has made available a great option for mass synthesis of malaria antigens, which otherwise could be prohibitive if it were to depend on the use of human serum and cells. The translation of feasibility to reality of production of a practical human malaria vaccine will depend on the definition of a safe and effective antigen.
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