Identification of differentially expressed genes using RNA fingerprinting by arbitrarily primed polymerase chain reaction.
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Animals↗
Biomedical subjects
F Mathieu-Daudé
Publications and source records attributed to F Mathieu-Daudé.
Reduced complexity probes for DNA arrays.
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DNA↗
Differentially expressed genes in the Trypanosoma brucei life cycle identified by RNA fingerprinting.
RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify genes that were differentially expressed during the life cycle of Trypanosoma brucei, as well as in response to heat shock. The standard RAP-PCR protocol was varied in two ways. First, the PCR reactions sometimes included a primer derived from the 5' mini-exon sequence, to ensure that most of the products contained the 5' end of mRNAs. Second, differentially amplified products were reamplified, isolated on single strand conformation polymorphism (SSCP) gels, cloned, and sequenced. Clones representing 32 different expressed sequence tags (ESTs) were obtained. Twenty-four ESTs were confirmed as differentially expressed by RT-PCR between different stages of the parasite cycle, or in response to temperature elevation. Nine clones had significant similarities to sequences already in the database. These transcripts included genes encoding cell surface proteins, metabolic enzymes, and heat shock proteins, either from trypanosomes or other organisms. Of particular interest, ESAG1 was shown to be heat-inducible in the procyclic stage. Most of the transcripts were unrelated to any other sequences in the database, and were deposited as new ESTs. The identification of stage-specific and heat shock-regulated transcripts will complement the growing T. brucei database. In addition, this experimental approach allows previous entries in the sequence database to be annotated with regulatory information.
Animals↗
Estrogen-responsive RING finger mRNA induction in gastrointestinal carcinoma cells following bile acid treatment.
The underlying molecular mechanisms of the tumor-promoting activity of bile acids such as chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) and the protective effect of ursodeoxycholic acid (UDCA) remain largely unclear. Using RNA arbitrarily primed PCR (RAP-PCR) for differential display, we identified, cloned and sequenced differentially expressed transcripts after treating gastric carcinoma cells (St 23132) with the bile acids CDCA, DCA and UDCA. One of these transcripts was identified to be an estrogen-responsive RING finger protein (efp) mRNA. The differential expression of efp in gastric cancer cells was confirmed by low stringency RT-PCR. efp mRNA levels were induced 3-fold in gastric carcinoma cells after CDCA and DCA treatment, whereas no change in expression was detected after UDCA treatment. Finally, treatment of the colon carcinoma cell line HT 29 with DCA resulted in a 2- to 5-fold induction of efp mRNA levels whereas UDCA did not induce efp. As expected, efp expression was also increased after 24 h of estrogen treatment. In summary, a synergy or a common pathway of tumor enhancement of bile acids and estrogen via efp in gastrointestinal carcinogenesis can be envisioned.
Bile Acids and Salts↗
DNA rehybridization during PCR: the 'Cot effect' and its consequences.
The rate of amplification of abundant PCR products generally declines faster than that of less abundant products in the same tube in the later cycles of PCR. As a consequence, differences in product abundance diminish as the number of PCR cycles increases. Rehybridization of PCR products which may interfere with primer binding or extension can explain this significant feature in late cycles. Rehybridization occurs with a half-time dependent on the reciprocal of the DNA concentration. Thus, if multiple PCR products are amplified in the same tube, reannealing occurs faster for the more abundant PCR products. In RT-PCR using an internal control, this results in a systematic bias against the more abundant of the two PCR products. In RNA fingerprinting by arbitrarily primed PCR (or differentially display of cDNAs), very large or absolute differences in the expression of a transcript between samples are preserved but smaller real differences may be gradually erased as the PCR reaction proceeds. Thus, this 'Cot effect' may systematically cause an underestimate of the true difference in starting template concentrations. However, differences in starting template concentrations will be better preserved in the less abundant PCR products. Furthermore, the slow down in amplification of abundant products will allow these rarer products to become more visible in the fingerprint which may, in turn, allow rarer cDNAs to be sampled more efficiently. In some applications, where the object is to stochiometrically amplify a mixture of nucleic acids, the bias against abundant PCR products can be partly overcome by limiting the number of PCR cycles and, thus, the concentration of the products. In other cases, abundance normalization at later cycles may be useful, such as in the production of normalized libraries.
Animals↗
Screening of differentially amplified cDNA products from RNA arbitrarily primed PCR fingerprints using single strand conformation polymorphism (SSCP) gels.
Arbitrarily primed PCR fingerprinting of RNA and differential display resolved on an acrylamide gel has been extensively used to detect differentially expressed RNAs. However, after a differentially amplified product is detected the next steps are labor-intensive: a small portion of the fingerprinting gel that contains the differentially amplified product is cut out, reamplified and the correct product is determined, typically by cloning and sequencing what is often a mixture of products of similar size. Here we use a native acrylamide gel to separate DNAs in the reamplified mixture based on single-stranded conformation polymorphisms. Reamplifications are performed for the region carrying the differentially amplified product and a corresponding region from an adjacent lane where the product is less prominent or not visible. Denaturation of the reamplified DNA followed by side-by-side comparison on an SSCP gel allows the classification of reamplified material into (i) those that can be directly cloned because the differentially amplified product is relatively pure, (ii) those that need to be reamplified from the SSCP gel before cloning and (iii) those that are too complex for further study. This screen should save considerable effort now wasted on directly cloning unsuitable products from RNA fingerprinting experiments. An example is presented of cloning a gene differentially expressed in Trypanosoma brucei life cycle.
Acrylic Resins↗
Genetic diversity and population structure of Trypanosoma brucei: clonality versus sexuality.
Genomic fingerprinting by arbitrarily primed PCR was used to analyze the genetic variability among 59 Trypanosoma brucei stocks representing the three T. brucei subspecies isolated from various hosts and different countries in Africa. 14 oligonucleotide primers revealed 355 polymorphic binary characters which were used for phenetic and phylogenetic analysis and to perform recombination tests exploring the linkage disequilibrium in the sample. There was good concordance between arbitrarily primed PCR polymorphisms and isoenzyme data previously collected for many of the same strains [1]. However, the arbitrarily primed PCR typing was more discerning than multilocus enzyme electrophoresis typing. Phenetic and phylogenetic analysis using arbitrarily primed PCR markers did not confirm T. brucei brucei and T. brucei rhodesiense as separate subspecies, but T. brucei gambiense group I was monophyletic, confirming this group as suitable for the subspecies status. With this exception, there were no clear lineages among the sample, other than clustering of East African stocks and clustering of West African stocks. Some features of the phylogenetic analysis suggested that the population structure was not strictly clonal though recombination tests showed linkage disequilibrium, even in the absence of repeated genotypes. While genotypes appear stable enough for tracking in applied studies, sexuality will impact at the evolutionary time scale, and may be more frequent under some ecological conditions. The arbitrarily primed PCR approach should be an effective and simple approach to follow epidemics and to quantify the role of sexuality in T. brucei populations.
Africa↗
Isozyme variability of Trypanosoma brucei s.l.: genetic, taxonomic, and epidemiological significance.
Seventy-eight Trypanosoma brucei s.l. stocks from different hosts, representing the three Trypanosoma brucei subspecies and three Trypanosoma evansi stocks, were studied for variation at 18 polymorphic isozyme loci. The results were used to determine the genetic variability among stocks and to estimate gene flow among populations. Total genetic variability in T. brucei s.l. was less than that in Trypanosoma cruzi, the agent of Chagas' disease. Results support a clonal population structure in T. brucei, but do not preclude a hypothesis of occasional mating. However, some natural clones of T. brucei s.l. appear as genetically stable and should be considered as useful taxonomic units in applied studies. Greater genotypic diversity was observed in trypanosomes isolated from wild mammals. Numerical taxonomy methods identified a group of clones representing most of the human stocks from Central and West Africa. This group probably corresponds to Trypanosoma brucei gambiense "group I" (Gibson, Parasitology Today 2, 255-257, 1986). As reported elsewhere, genetic evidence of the subspecies Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense was ambiguous, suggesting that these taxa represent more "nosodemes" rather than actual genetic clades.
Alleles↗
Mixed populations of Trypanosoma brucei in wild Glossina palpalis palpalis.
In many previous characterization studies of Trypanozoon, isolates have been subpassaged numerous times in laboratory rodents until a quantity of trypanosomes sufficient for analysis has been obtained. In addition to the numerous biochemical effects of such a process on the parasite, it appears probable that adaptation to an unnatural host may also serve to filter out less virulent populations from mixed infections, leading to an underestimate of the true level of genetic diversity. By the early cloning of trypanosomes from susceptible captive flies infected from the primary isolate--the midgut of a wild tsetse--the present study provides evidence of the range of genetically different Trypanosoma brucei populations which may coexist within the midgut of individual tsetse flies in nature. The three primary isolates from tsetse yielded one, five and nine genetically distinct populations. Cloned populations were confirmed as T. brucei using the polymerase chain reaction, and were characterized by karyotype analysis and multilocus isoenzyme electrophoresis. These data allowed a limited assessment of the level of genetic variability in natural populations of T. brucei.
Animals↗
Isoenzyme electrophoresis for parasite characterization.
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Alleles↗
Multilocus isozyme identification of Trypanosoma brucei stocks isolated in central Africa: evidence for an animal reservoir of sleeping sickness in Congo.
Six Congolese and 3 Zairian Trypanosoma brucei stocks were studied by isozyme cellulose acetate electrophoresis. Twenty isozyme systems were used, of which only 5 showed variability. These 5 polymorphic systems made it possible to identify 5 different zymodemes. Zymodemes isolated from man were recorded both from pig and sheep too, which confirms the results of previous authors. This favors the existence of an animal reservoir of human African trypanosomiasis in the Congo, which could play a role in the transmission of the disease, at least by the maintenance of residual foci.
Animals↗
Chromosome rearrangement in Leishmania mexicana M379.
Circular extrachromosomal elements were observed in a variety of Leishmania species. We show here that two lines originating from the same isolate have been found to contain a circular DNA molecule of 26.6 kb and a linear chromosome of about 250 kb, respectively, which share a homology of more than 20 kb. The circular DNA molecule and its related region on the linear chromosome were cloned and their restriction maps compared. This investigation reveals information about chromosome rearrangement in L. mexicana M379. Further examination will enable us to understand the nature of chromosome rearrangement such as circularization or linearization.
Animals↗