Excision of Ds produces waxy proteins with a range of enzymatic activities.

The waxy (wx) locus of maize encodes an enzyme responsible for the synthesis of amylose in endosperm tissue. The phenotype of the Dissociation (Ds) insertion mutant wx-m1 is characterized by endosperm sectors that contain different levels of amylose. We have cloned the Wx gene from this allele and from two germinal derivatives, S5 and S9, that produce intermediate levels of amylose. The Ds insertion in wx-m1 is in exon sequences, is 409 bp in length and represents an example of a class of Ds elements that are not deletion derivatives of the Activator (Ac) controlling element. The two germinal derivatives, S5 and S9, lack the Ds element but contain an additional 9 and 6 bp, respectively, at the site of Ds insertion. The level of Wx mRNA and Wx protein in S5 and S9 is essentially the same as in normal endosperm tissue but Wx enzymatic activity is reduced. Thus, the lesions in S5 and S9 lead to the addition of amino acids in the Wx protein, resulting in Wx enzymes with altered specific activities. This work supports the notion that the maize transposable elements may serve a function in natural populations to generate genetic diversity, in this case, proteins with new enzymatic properties.     

Diversification of the rice Waxy gene by insertion of mobile DNA elements into introns.

The waxy (wx) gene of Oryza glaberrima was cloned, and its nucleotide sequence was determined. A waxy mutant of O. glaberrima showing a glutinous phenotype was found to contain a substitution mutation generating a termination codon in the coding region of the wx gene. The Wx sequence of O. glaberrima was different from that of Oryza sativa by substitutions and insertions/deletions, among which only a few substitutions occurred in several exons not to severely alter the amino acid sequence of the Wx protein. The most striking difference observed in introns was a 139-bp deletion (or insertion) in intron 10 of O. glaberrima (or O. sativa). In O. sativa, 125 bp of the 139-bp sequence was flanked by direct repeats of a 14-bp sequence. A sequence homologous to the 125-bp sequence was found in the region preceding exon 2; this sequence was also flanked by direct repeats of another 14-bp sequence. This result and the observation that the 125-bp sequence was interspersed in rice genomes indicate that they are SINEs (short interspersed elements) in the plant system. We also identified a DNA sequence with long terminal inverted repeats in intron 13 of both O. glaberrima and O. sativa. This sequence was present in multiple copies in rice genomes, suggesting that it is a transposable element. These results obtained suggest that mobile DNA elements have diversified the rice Waxy gene by inserting into introns, each of which may originally have a length of about 100 bp.     

Nucleotide sequence of the maize transposable element Mul

A cloned DNA fragment from the maize allele Adhl-S3034 contains all of Mul, an insertion element involved in Robertson's Mutator activity. The element is 1367 base pairs (bp) long and is flanked by nine bp direct repeats of insertion site DNA. It has inverted terminal repeats of 215 and 213 bp showing 95% homology. Within the element are two direct repeats of 104 bp showing 96% homology. Four open reading frames (ORFs) were found, two in each DNA strand. Mul can be divided into two halves, each containing one terminal inverted repeat, an internal direct repeat, and two overlapping ORFs. The GC content of each half is high (70%), while that of a central 60 base portion of the element is low (26%). The central region contains the only sequence resembling the TAATA Goldberg and Hogness eukaryotic promoter signal. Multiple copies of DNA sequences related to Mul found in Mutator maize plants are generally similar in organization to the cloned element. A larger version containing a discrete 300 to 400 base pair insertion was found in some Mutator lines.     

Analysis of sh-m6233, a mutation induced by the transposable element Ds in the sucrose synthase gene of Zea mays

The unstable allele sh-m6233 caused by insertion of the transposable element Ds into the sucrose synthase gene of maize, was cloned. The mutation is caused by the insertion of an ˜4 kb DNA segment, consisting of two identical Ds elements of ˜2000 bp length, of which one is inserted into the center of the other in inverted orientation. This structure is, at the level of restriction mapping and partial DNA sequencing, identical to the double Ds element found in a larger insert in the mutant allele sh-m5933. 8 bp of host DNA are duplicated upon insertion. In a revertant, a 6-bp duplication is retained.     

Characterization of the IS895 family of insertion sequences from the cyanobacterium Anabaena sp. strain PCC 7120.

A family of repetitive elements from the cyanobacterium Anabaena sp. strain PCC 7120 was identified through the proximity of one element to the psbAI gene. Four members of this seven-member family were isolated and shown to have structures characteristic of bacterial insertion sequences. Each element is approximately 1,200 bp in length, is delimited by a 30-bp inverted repeat, and contains two open reading frames in tandem on the same DNA strand. The four copies differ from each other by small insertions or deletions, some of which alter the open reading frames. By using a system designed to trap insertion elements, one of the elements, denoted IS895, was shown to be mobile. The target site was not duplicated upon insertion of the element. Two other filamentous cyanobacterial strains were also found to contain sequences homologous to IS895.     

Molecular Analysis of the Maize Wx-B3 Allele Indicates That Precise Excision of the Transposable Ac Element Is Rare

The somatic and germinal behavior of the maize wx-B3 mutation indicates that this Ac allele rarely reverts. Endosperms containing wx-B3 display tiny and infrequent Wx revertant sectors while no significant reversion is detected when wx-B3 pollen is stained with I/KI. Previous studies of other transposable element alleles that revert infrequently have implicated low levels of element excision. Unlike these other alleles, the wx-B3 Ac element is indistinguishable from fully active Ac elements with respect to its structure, and its ability to transpose from the Wx gene or to trans-activate a Ds element. Characterization of somatic and germinal excision events lead us to conclude that excision of the wx-B3 Ac element almost always produces null alleles. Furthermore, the excellent correlation between the position of the wx-B3 mutation on the physical and genetic maps indicates that the Ac insertion is the only lesion of wx-B3. As a result, precise excision of this Ac should restore Wx function. The fact that revertant sectors and pollen grains are rare indicates that precise excision of Ac is also rare. The finding that the wx-B3 reversion frequency is comparable whether wx-B3 is hemizygous or over a wx allele with a wild-type insertion site illustrates a fundamental difference between the excision mechanisms of Ac and Drosophila P elements.     

Maize Mitochondrial Plasmid S-1 Sequences Share Homology with Chloroplast Gene psbA

The linear, 6397-base pair (bp), mitochondrial S-1 DNA molecule from maize contains a 420-bp segment that is homologous with the chloroplast gene (psbA) that codes for the quinone binding protein of photosystem II. This is the first report of a chloroplast sequence in a naturally occurring viral-like or plasmid DNA. The complete sequence of the S-1 chloroplast segment has been compared with homologous regions of six different chloroplast genes. The S-1 segment has diverged from the other genes both by length mutation and base substitution. Several of the length mutations are exact adjacent tandem duplications of 4 and 5 bp similar to "footprints" left after excision of transposable elements in maize nuclear DNA.     

Molecular analysis of theBg-rbg transposable element system ofZea mays L.

Summary The two components of theBg-rbg transposable element system of maize have been cloned. TheBg element, isolated from the mutable allelewx-m32 :: Bg is inserted in the intron of theWaxy (Wx) gene between exons 12 and 13. The length of the element is of 4869 bp.Bg has 5 by terminal inverted repeats, and generates upon insertion an 8 by direct duplication of the target sequence. Both ends of theBg element contain a 76 by direct repeat adjacent to the terminal inverted repeats. The hexamer motif TATCG_kC ^G is here repeated several times in direct or inverse orientation. Therbg element was isolated from the mutable alleleo2m(r) where it is located in the promoter region of theOpaque-2 (O2) gene.rbg is approximately 4.5 kb in length, has terminal inverted repeats identical to those of theBg element, and is also flanked by an 8 by direct duplication at the target site. LikeBg, rbg carries the 76 by direct repeats. Restriction enzyme analysis reveals that, compared toBg, the receptor element is distinguishable by small deletion and insertion events. Sequence data indicate that not more than 75% homology exists at the DNA level between therbg element and the autonomousBg element.     

A transposon-like sequence with short terminal inverted repeats in the nuclear genome of Chlamydomonas reinhardtii

A 1.2 kb DNA sequence, flanked by a potential seven base target-site duplication, was found inserted into a TOC1 transposable element from Chlamydomonas reinhardtii. The insertion sequence, named TOC2, is a member of a family of repeated DNA sequences that is present in all the C. reinhardtii strains tested. It resembles class II transposable elements: it possesses short 14 bp imperfect terminal repeats that begin AGGAGGGT, and sub-terminal direct repeats located within 250 bp of the termini. No large open reading frames were found. The terminal bases and length of target-site duplication are important in classifying transposable elements. On this basis TOC2 does not fall readily into existing families of class II transposable elements found in plants.     

Nucleotide sequence of IS110, an insertion sequence of Streptomyces coelicolor A3(2).

The nucleotide sequences of the Streptomyces transposable element IS110 and its insertion site in the DNA of a derivative of the temperate phage luminal diameter C31 were determined. The element is inserted about 460 bp from the right-hand end of luminal diameter C31 DNA, in a region of apparently non-coding DNA. The target site (in a run of seven C residues) is within an 11 bp sequence homologous with one end of IS110. The inserted element is flanked by runs of 11 and 15 C residues which form part of more extensive regions of homology between the left and right junction regions. Imperfect inverted repeats (10 matches out of 15 bp) are present near (but not at) the ends of IS110. The whole IS110 element contains about 1550 bp of which 71% are G-C bp. One major potentially protein-coding region (ORF 1215) was detected, of 1215 bp, the product of which, a presumptively soluble protein of MR 43,563, was not overtly related to any entry in a protein sequence database. A smaller open reading frame (ORF 330) was tentatively identified in the opposite strand of the ORF 1215 region.     

Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system.

Plasmids containing heteroallelic copies of the Saccharomyces cerevisiae HIS3 gene undergo intramolecular gene conversion in mitotically dividing S. cerevisiae cells. We have used this plasmid system to determine the minimum amount of homology required for gene conversion, to examine how conversion tract lengths are affected by limited homology, and to analyze the role of flanking DNA sequences on the pattern of exchange. Plasmids with homologous sequences greater than 2 kilobases have mitotic exchange rates as high as 2 x 10(-3) events per cell per generation. As the homology is reduced, the exchange rate decreases dramatically. A plasmid with 26 base pairs (bp) of homology undergoes gene conversion at a rate of approximately 1 x 10(-10) events per cell per generation. These studies have also shown that an 8-bp insertion mutation 13 bp from a border between homologous and nonhomologous sequences undergoes conversion, but that a similar 8-bp insertion 5 bp from a border does not. Examination of independent conversion events which occurred in plasmids with heteroallelic copies of the HIS3 gene shows that markers within 280 bp of a border between homologous and nonhomologous sequences undergo conversion less frequently than the same markers within a more extensive homologous sequence. Thus, proximity to a border between homologous and nonhomologous sequences shortens the conversion tract length.     

Allelic variation of the Waxy gene in foxtail millet [Setaria italica (L.) P. Beauv.] by single nucleotide polymorphisms

The Waxy (Wx) gene product controls the formation of a straight chain polymer of amylose in the starch pathway. Dominance/recessiveness of the Wx allele is associated with amylose content, leading to non-waxy/waxy phenotypes. For a total of 113 foxtail millet accessions, agronomic traits and the molecular differences of the Wx gene were surveyed to evaluate genetic diversities. Molecular types were associated with phenotypes determined by four specific primer sets (non-waxy, Type I; low amylose, Type VI; waxy, Type IV or V). Additionally, the insertion of transposable element in waxy was confirmed by ex1/TSI2R, TSI2F/ex2, ex2int2/TSI7R and TSI7F/ex4r. Seventeen single nucleotide polymorphims (SNPs) were observed from non-coding regions, while three SNPs from coding regions were non-synonymous. Interestingly, the phenotype of No. 88 was still non-waxy, although seven nucleotides (AATTGGT) insertion at 2,993 bp led to 78 amino acids shorter. The rapid decline of r ² in the sequenced region (exon 1–intron 1–exon 2) suggested a low level of linkage disequilibrium and limited haplotype structure. K _s values and estimation of evolutionary events indicate early divergence of S. italica among cereal crops. This study suggested the Wx gene was one of the targets in the selection process during domestication. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparison of the virulence plasmid genomes of two strains of Shigella which lost the ability to bind Congo red

We determined and analyzed the Shigella flexneri serotype 5 (pSF5) and S. dysenteriae serotype 1 (pSD1) virulence plasmid genomes. The total length of pSF5 is 136513 bp, including 165 open reading frames (ORFs). Of these ORFs, 133 were identified and 32 of those had no significant homology to proteins with known functions. The length of pSD1 is 182545 bp, including 224 ORFs, of which we identified 181. The remaining 43 ORFs were not significantly homologous to proteins with known functions. The insertion sequence (IS) elements are 53787 bp in pSF5, and 49616 bp in pSD1, which represents 39.4% and 27.1% of the genome, respectively. There are 22 IS element types in pSF5 and pSD1, among which we report ISEc8 and ISSbo6 for the first time in the Shigella virulence plasmid. Compared to pCP301, there are a large number of deleted genes and gene inversions in both pSF5 and pSD1. The ipa-mxi-spa locus in pSF5 is completely absent, and the genes related to the O-antigen biosynthesis are partially missing. In contrast, the above genes in pSD1 are integral, with the exception of virF. The whole genome analysis of the two plasmids shows that the loss of genes related to gene invasion or regulation also obliterates the ability of pPF5 and pSD1 to bind Congo red (Crb). Whether these genes determine the Crb function requires continued investigation. These authors contributed equally to this work.