Biodiversita' Ed Evoluzione
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Alma Mater Studiorum – Università di Bologna DOTTORATO DI RICERCA IN BIODIVERSITA' ED EVOLUZIONE Ciclo XXVIII Settore Concorsuale di afferenza: 05/B1 – Zoologia ed Antropologia Settore Scientifico disciplinare: BIO/05 – Zoologia TRANSPOSABLE ELEMENTS IN ARTHROPODS GENOMES WITH NON-CANONICAL REPRODUCTIVE STRATEGIES. Presentata da: Dot.ssa Claudia Scavariello Coordinatore Dottorato Relatore Prof.ssa Barbara Mantovani Prof.ssa Barbara Mantovani Correlatore Dott. Andrea Luchetti Esame finale anno 2016 INDEX 1. INTRODUCTION-------------------------------------------------------------------------------------------------------------------------------------1 1.1. History of transposable elements--------------------------------------------------------------------------------1 1.1.1 TEs Classification---------------------------------------------------------------------------------------------------3 1.2. R2 non-LTR retrotransposon----------------------------------------------------------------------------------------------6 1.2.1. R2 history and structure--------------------------------------------------------------------------------------6 1.2.2. R2 retrotranscription mechanism and ribozyme structure--------------10 1.3. TEs survival and the host genome--------------------------------------------------------------------------------12 1.3.1. The impact of TEs on eukaryotic genomes---------------------------------------------12 1.3.2. TEs transposition rate dynamics and host reproductive strategies------------------------------------------------------------------------------------------------------------------14 1.3.3. Site-Specific insertions--------------------------------------------------------------------------------------16 1.3.4. Horizontal transfer-----------------------------------------------------------------------------------------------17 1.4. The order Phasmida---------------------------------------------------------------------------------------------------------20 1.4.1. The Bacillus genus----------------------------------------------------------------------------------------------21 1.4.2. Reproductive strategies------------------------------------------------------------------------------------23 1.4.3. Phylogenetic relationships------------------------------------------------------------------------------25 1.5. The order Notostraca (Branchiopoda, Crustacea, Arthropoda)-----------------27 1.5.1. Triops genus Reproduction-----------------------------------------------------------------------------28 1.5.2. The difficult phylogeny of the order Notostraca-----------------------------------30 2. RESEARCH AIMS------------------------------------------------------------------------------------------------------------------------------33 2.1.Main results in Bacillus genus------------------------------------------------------------------------------------------34 2.1.1. Evolutionary dynamics of R2 retroelement in the genome of bisexual and parthenogenetic Bacillus rossius populations--------35 2.1.2. Degenerate R2 Element Replication and rDNA Genomic Turnover in the Bacillus rossius Stick Insect--------------------------------------------------------------54 2.1.3. First case of R2 non-LTR retrotransposon horizontal transfer------73 2.2. Main results in Triops cancriformis------------------------------------------------------------------------------93 2.2.1. TEs content and composition in Triops cancriformis-------------------------93 3. ABROAD PERIOD------------------------------------------------------------------------------------------------------------------------------97 4. CONCLUSIONS------------------------------------------------------------------------------------------------------------------------------------99 5. REFERENCES-----------------------------------------------------------------------------------------------------------------------------------105 1. INTRODUCTION 1.1. History of transposable elements Until the half of the 20th century, the genome was basically considered like a pearl necklace with genes ordered on chromosomes, behaving as stable and almost fixed entities evolving through the accumulation of random mutations at slow rate. Only in the late ’40s, Barbara McClintock was able to demonstrate that genomes are highly dynamic entities thanks to her discovery that some genes could be able to move from a chromosomal site to another one. Genetic studies on maize led McClintock to characterize a DNA sequence that could move from one chromosome location to another causing chromosomal breakage or instability, and leading to chromosomal rearrangements. She also found that in one maize line the breakage on the short arm of chromosome 9 was recurrent. She assumed that these events were from the product of a particular unstable genetic element named Ds (i.e., Dissociation). Through genetic crosses and cytological observations, she understood that the instability of Ds elements was dependent on the presence of another element type designated as Ac for Activator. Her data on the first transposable elements (TEs) ever found, Ac and Ds, were reported in “The origin and behavior of mutable loci in maize”, article published in 1950 (McClintock, 1950). In the 1980s, it was understood that Ac and Ds are autonomous and non- autonomous elements and only Ac encodes the functional transposase enzyme required for the mobility of both elements (McClintock, 1953). Only twenty years later McClintock's discoveries, the scientific community started to acknowledge her assumptions, thanks to the work of the American microbiologist James A. Shapiro, the American geneticist Margaret Kidwell and French biologist Georges Picard. Shapiro worked on the galactose (gal) operon in E. coli. He isolated a gal-mutants in the late 1960s and he demonstrated that the mutations were caused by the recurrent insertions of the same long segment of DNA, called insertion sequence 1 (IS1). All IS1 were very similar in structure: they had a single gene sequence encoding a transposase enzyme and delimited by short terminal inverted repeats (TIRs) (Shapiro 1969). Kidwell and Picard discovered the presence of P elements in the Drosophila melanogaster genome through studies on hybrid dysgenesis. In the hybrids of crosses between females from laboratory stocks with males from specific natural populations (called P strains), dysgenesis induced frequent sterility and genetic instability, but curiously the same situation was not observed in the reciprocal crosses (Kidwell 1977). The molecular characterization of mutations in these hybrids established that hybrid dysgenesis was 1 caused by a mobile element activated during dysgenic crosses: this P element was consistently absent from laboratory stocks but present in most wild-caught flies (Picard et al. 1978; Kidwell 1979). Like IS elements, P elements have a short TIRs and a single gene encoding a transposase. In the early 1980s, studies on another popular model organism, the yeast Saccharomyces cerevisiae, revealed the presence of TEs, the so-called Ty elements, completely different from other mobile elements known at that time. These elements showed long terminal repeats (LTRs) flanking an array of coding sequences and some common characteristics with retroviral proviruses, such as a reverse transcriptase domain. An American geneticist, Jef Boeke, confirmed that Ty elements transpose through an RNA intermediate which is converted back into a DNA molecule for its integration in the yeast genome. Boeke coined the term retrotransposon to describe TEs that move via this process (Boeke et al. 1985). In the 1980s, further, appropriate molecular tools were developed for eukaryotic systems and many non-model organisms started to be investigated, highlighting that TEs were universal components of all living organisms. During those years, numerous empirical data accumulated about TEs structure and transposition mechanisms but little had been speculated on theoretical expectations. At that time, TEs studies were influenced by two different theories trying to explain the ubiquitous presence of TEs as well as their high genomic proportions. The first theory that influenced TEs studies was the neutral theory at the end of the 1970s and early 1980s, of whom Dawkins and Crick were the main leaders. This theory considered these elements as “junk’’ and ‘‘selfish’’ pieces of DNA whose only “function” was self-preservation (Dawkins, 1976; Crick, 1979; Doolittle and Sapienza, 1980). In his book The Selfish Gene Richard Dawkins (1976) suggested the idea that surplus non-coding DNA in eukaryotic genomes was selfish. Selfish DNA is a DNA sequence with the unique apparent function to replicates itself, also exploiting the host molecular mechanisms, this is why it was considered a ‘molecular parasite’ in the selfish DNA theory. The presence of selfish DNA can be explained by the inability of the organism to efficiently prevent its increase in the genome. Another theory explained instead the large presence of TEs in living organisms as due to some advantage they perform for their host genomes. The “phenotypic paradigm” of the neo-Darwinian theory speculated that non-transcribed DNA, and in particular repetitive sequences, were necessary as regulators or somehow essential to chromosome structures or pairing. Under this view, this genomic portion would facilitate genetic rearrangements increasing the possibility of genome evolution (long-term phenotypic 2 benefit); or it could evolve into sequences with new functions (McDonald,