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Category: Microbial Genetics and Molecular Biology
I Elements in Drosophila melanogaster, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555817954/9781555812096_Chap33-1.gif /docserver/preview/fulltext/10.1128/9781555817954/9781555812096_Chap33-2.gifAbstract:
At the beginning of the 1970s Picard and L’Héritier reported that crosses between particular strains of Drosophila melanogaster produce progeny exhibiting genetic abnormalities. In 1976 Picard reported that the factor responsible for IR hybrid dysgenesis is a transposable element, the first discovered in Drosophila, and named it the I factor. The determination of its sequence showed that it belongs to the class of non-long terminal repeat retrotransposons (NLRs) also known as long interspersed nucleotidic elements (LINEs). The I factor is one of the models used to study the mechanism of transposition of NLRs because it can be mobilized at high frequency by dysgenic crosses, giving the opportunity to study the molecular mechanism of transposition in vivo. The D. melanogaster species can be divided into two classes of strains according to the IR system of hybrid dysgenesis, inducer (or I) and reactive (or R). I strains contain several complete and functional I factors, R strains do not. The mechanism of transposition of the I factor is thought to be related to target-primed reverse transcription (TPRT) requiring a full-length RNA intermediate. The study of deletion derivatives indicates that the sequences comprised between nucleotides 41 and 100 might be involved in the inhibition of somatic expression. The study of deletion derivatives showed that more than one region of the protein is involved in DNA binding and that the cysteine-rich motifs are not essential for this, but are required for the formation of the high molecular weight structures.
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Results of crosses between the two categories of strains involved in the IR system of hybrid dysgenesis.
Results of crosses between the two categories of strains involved in the IR system of hybrid dysgenesis.
Structure of the I factor and of its RNA. Boxes represent the two ORFs. C represents cysteine-rich motifs, and EN, RT, and RH represent the endonuclease, reverse transcriptase, and RNase H domains, respectively. Below is shown the RNA of the I factor starting at the first nucleotide of the element and ending after the UAA repeats at the 3′end (double line).
Structure of the I factor and of its RNA. Boxes represent the two ORFs. C represents cysteine-rich motifs, and EN, RT, and RH represent the endonuclease, reverse transcriptase, and RNase H domains, respectively. Below is shown the RNA of the I factor starting at the first nucleotide of the element and ending after the UAA repeats at the 3′end (double line).
Retrotransposition of the I factor generating repeats at the 3′end. The endonuclease encoded by the I factor makes staggered nicks in chromosomal DNA (A). The reverse transcriptase of the element associated with the transposition intermediate, which extends beyond the UAA repeats, binds the target site and uses the 3′-OH at the end of chromosomal DNA to initiate reverse transcription (B). After polymerization of a few nucleotides (C) the RNA may dissociate and reassociate to a short complementary sequence in the newly synthesized cDNA (D). Reverse transcriptase proceeds to the 5′end of the RNA template and switches to the target DNA (E). After degradation of the RNA by the I factor RNase H, synthesis of the second strand of the cDNA, and ligation, there is insertion of a full-length I element flanked by a target site duplication. The target site is shown in regular type, the RNA is shown in boldface (except for the extra nucleotides downstream of the UAA repeats, which are in italics [NNNN]), and the cDNA is shown in bold italics. The duplication of the target sequence is underlined. Lowercase letters in panels E and F indicate newly synthesized DNA of the target site duplication.
Retrotransposition of the I factor generating repeats at the 3′end. The endonuclease encoded by the I factor makes staggered nicks in chromosomal DNA (A). The reverse transcriptase of the element associated with the transposition intermediate, which extends beyond the UAA repeats, binds the target site and uses the 3′-OH at the end of chromosomal DNA to initiate reverse transcription (B). After polymerization of a few nucleotides (C) the RNA may dissociate and reassociate to a short complementary sequence in the newly synthesized cDNA (D). Reverse transcriptase proceeds to the 5′end of the RNA template and switches to the target DNA (E). After degradation of the RNA by the I factor RNase H, synthesis of the second strand of the cDNA, and ligation, there is insertion of a full-length I element flanked by a target site duplication. The target site is shown in regular type, the RNA is shown in boldface (except for the extra nucleotides downstream of the UAA repeats, which are in italics [NNNN]), and the cDNA is shown in bold italics. The duplication of the target sequence is underlined. Lowercase letters in panels E and F indicate newly synthesized DNA of the target site duplication.
Ability to repress I factor activity in "reconstructed" R stocks devoid of transposed I elements obtained in the progeny of RSF females. (A) Experimental scheme. RSF females were backcrossed to R males, and female and male progeny having the complete genotype of an R strain and devoid of transposed copies of the I factor were selected to establish the reconstructed R stocks. I factor activity was estimated by crossing at each generation females of the stocks to I males and determining the hatching percentages of the eggs laid by their daughters (SF females). I and R represent half genomes from an I or an R strain, respectively. (B) Ability over generations of the females of the reconstructed R stocks to repress I factor activity. This ability is high during the first generations (high fertility of their SF daughters) and decreases progressively generation after generation (low fertility of the SF daughters). The frequency of transposition of I factors is higher in more sterile SF females ( 88 ). Data are from reference 17 .
Ability to repress I factor activity in "reconstructed" R stocks devoid of transposed I elements obtained in the progeny of RSF females. (A) Experimental scheme. RSF females were backcrossed to R males, and female and male progeny having the complete genotype of an R strain and devoid of transposed copies of the I factor were selected to establish the reconstructed R stocks. I factor activity was estimated by crossing at each generation females of the stocks to I males and determining the hatching percentages of the eggs laid by their daughters (SF females). I and R represent half genomes from an I or an R strain, respectively. (B) Ability over generations of the females of the reconstructed R stocks to repress I factor activity. This ability is high during the first generations (high fertility of their SF daughters) and decreases progressively generation after generation (low fertility of the SF daughters). The frequency of transposition of I factors is higher in more sterile SF females ( 88 ). Data are from reference 17 .
Modifications of reactivity over generations induced by aging. A strongly reactive strain was maintained by producing each generation from either young-laying females (Y) or old-laying females (O). The reactivity level was determined at various generations by measuring the hatching percentages of the eggs laid by SF females obtained by crossing females of these stocks with I males. Reactivity remained strong in the Y stock but became progressively weaker generation after generation in the O stock. This change in reactivity is reversible as shown when young-laying females are used again (R1 and R2). The frequency of transposition of I factors is higher when SF females are derived from strongly reactive females than when derived from weakly reactive females ( 88 ). Modified from reference 14 with permission.
Modifications of reactivity over generations induced by aging. A strongly reactive strain was maintained by producing each generation from either young-laying females (Y) or old-laying females (O). The reactivity level was determined at various generations by measuring the hatching percentages of the eggs laid by SF females obtained by crossing females of these stocks with I males. Reactivity remained strong in the Y stock but became progressively weaker generation after generation in the O stock. This change in reactivity is reversible as shown when young-laying females are used again (R1 and R2). The frequency of transposition of I factors is higher when SF females are derived from strongly reactive females than when derived from weakly reactive females ( 88 ). Modified from reference 14 with permission.
Distribution of I elements in the eight species of the D. melanogaster subgroup. The phylogenetic relationships between the eight species of the subgroup are drawn according to reference 55. The number of plus signs indicates the intensity of the signals observed in Southern blot experiments (21).
Distribution of I elements in the eight species of the D. melanogaster subgroup. The phylogenetic relationships between the eight species of the subgroup are drawn according to reference 55. The number of plus signs indicates the intensity of the signals observed in Southern blot experiments (21).