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S R Wessler

Publications and source records attributed to S R Wessler.

At least 19 recordsLinked to original sources
PubMed2002

Evaluation of Hbr (MITE) markers for assessment of genetic relationships among maize ( Zea mays L.) inbred lines.

Recently, a new type of molecular marker has been developed that is based on the presence or absence of the miniature inverted repeat transposable element (MITE) family Heartbreaker ( Hbr) in the maize genome. These so-called Hbr markers have been shown to be stable, highly polymorphic, easily mapped, and evenly distributed throughout the maize genome. In this work, we used Hbr-derived markers for genetic characterization of a set of maize inbred lines belonging to Stiff Stalk (SS) and Non-Stiff Stalk (NSS) heterotic groups. In total, 111 markers were evaluated across 62 SS and NSS lines. Seventy six markers (68%) were shared between the two groups, and 25 of the common markers occurred at fairly low frequency (</=0.20). Only two markers (3%) were monomorphic in all samples. Although DNA sequencing indicated that 5.5% of same-sized DNA fragments were non-homologous, this result did not affect the cluster analyses (i.e., relationships obtained from the Hbr data were congruent with those derived from pedigree information). Distance matrices generated from Hbr markers were significantly correlated ( p<0.001) with those obtained from pedigree ( r=0.782), RFLPs ( r=0.747), and SSRs ( r=0.719). Overall, these results indicated that Hbr markers could be used in conjunction with other molecular markers for genotyping and relationship studies of related maize inbred lines.

Journal Article↗
PubMed2001

P instability factor: an active maize transposon system associated with the amplification of Tourist-like MITEs and a new superfamily of transposases.

Miniature inverted-repeat transposable elements (MITEs) are widespread and abundant in both plant and animal genomes. Despite the discovery and characterization of many MITE families, their origin and transposition mechanism are still poorly understood, largely because MITEs are nonautonomous elements with no coding capacity. The starting point for this study was P instability factor (PIF), an active DNA transposable element family from maize that was first identified following multiple mutagenic insertions into exactly the same site in intron 2 of the maize anthocyanin regulatory gene R. In this study we report the isolation of a maize Tourist-like MITE family called miniature PIF (mPIF) that shares several features with PIF elements, including identical terminal inverted repeats, similar subterminal sequences, and an unusual but striking preference for an extended 9-bp target site. These shared features indicate that mPIF and PIF elements were amplified by the same or a closely related transposase. This transposase was identified through the isolation of several PIF elements and the identification of one element (called PIFa) that cosegregated with PIF activity. PIFa encodes a putative protein with homologs in Arabidopsis, rice, sorghum, nematodes, and a fungus. Our data suggest that PIFa and these PIF-like elements belong to a new eukaryotic DNA transposon superfamily that is distantly related to the bacterial IS5 group and are responsible for the origin and spread of Tourist-like MITEs.

Base Sequence↗
PubMed2001

Role of mRNA secondary structure in translational repression of the maize transcriptional activator Lc(1,2).

Lc, a member of the maize (Zea mays) R/B gene family, encodes a basic helix-loop-helix transcriptional activator of the anthocyanin biosynthetic pathway. It was previously shown that translation of the Lc mRNA is repressed by a 38-codon upstream open reading frame (uORF) in the 5' leader. In this study, we report that a potential hairpin structure near the 5'end of the Lc mRNA also represses downstream translation in the rabbit reticulocyte in vitro translation system and in transient transformation assays. Base pairing of the hairpin is important for repression because its destabilization increases translation of the uORF and the downstream ORF. However, translation of the uORF is not required for the hairpin-mediated repression. Instead, the uORF and the 5'-proximal hairpin mediate two independent levels of repression. Although the uORF represses downstream translation due to inefficient reinitiation of ribosomes that translate uORF, the hairpin inhibits ribosome loading at the 5' end of the mRNA.

Base Sequence↗
PubMed2001

Insertion preference of maize and rice miniature inverted repeat transposable elements as revealed by the analysis of nested elements.

A 128-bp insertion into the maize waxy-B2 allele led to the discovery of Tourist, a family of miniature inverted repeat transposable elements (MITEs). As a special category of nonautonomous elements, MITEs are distinguished by their high copy number, small size, and close association with plant genes. In maize, some Tourist elements (named Tourist-Zm) are present as adjacent or nested insertions. To determine whether the formation of multimers is a common feature of MITEs, we performed a more thorough survey, including an estimation of the proportion of multimers, with 30.2 Mb of publicly available rice genome sequence. Among the 6600 MITEs identified, >10% were present as multimers. The proportion of multimers differs for different MITE families. For some MITE families, a high frequency of self-insertions was found. The fact that all 340 multimers are unique indicates that the multimers are not capable of further amplification.

Base Sequence↗
PubMed2000

The MITE family heartbreaker (Hbr): molecular markers in maize.

Transposable elements are ubiquitous in plant genomes, where they frequently comprise the majority of genomic DNA. The maize genome, which is believed to be structurally representative of large plant genomes, contains single genes or small gene islands interspersed with much longer blocks of retrotransposons. Given this organization, it would be desirable to identify molecular markers preferentially located in genic regions. In this report, the features of a newly described family of miniature inverted repeat transposable elements (MITEs) (called Heartbreaker), including high copy number and polymorphism, stability, and preference for genic regions, have been exploited in the development of a class of molecular markers for maize. To this end, a modification of the AFLP procedure called transposon display was used to generate and display hundreds of genomic fragments anchored in Hbr elements. An average of 52 markers were amplified for each primer combination tested. In all, 213 polymorphic fragments were reliably scored and mapped in 100 recombinant inbred lines derived from a cross between the maize inbreds B73 x Mo17. In this mapping population, Hbr markers are distributed evenly across the 10 maize chromosomes. This procedure should be of general use in the development of markers for other MITE families in maize and in other plant and animal species where MITEs have been identified.

Base Sequence↗
PubMed2000

Recent, extensive, and preferential insertion of members of the miniature inverted-repeat transposable element family Heartbreaker into genic regions of maize.

A 314-bp DNA element called Heartbreaker-hm1 (Hbr-hm1) was previously identified in the 3' untranslated region of a mutant allele of the maize disease resistance gene HM1. This element has structural features of miniature inverted-repeat transposable elements (MITEs) and is a member of a large family of approximately 4,000 copies in the maize genome. Unlike previously described MITEs, most members of the Hbr family display over 90% sequence identity. This, coupled with the insertion of an Hbr element into an allele of the HM1 gene, suggested that this family might have spread recently throughout the genome. Consistent with this view is the finding that Hbr insertion sites are remarkably polymorphic. Ten of ten loci containing Hbr elements were found to be polymorphic for the presence or absence of Hbr among a collection of maize inbred lines and teosinte strains. Despite the fact that over 80% of the maize genome contain moderate to highly repetitive DNA, we find that randomly chosen Hbr elements are predominantly in single or low copy regions. Furthermore, when used to query both the public and private databases of plant genes, over 50% of the sequences flanking these Hbr elements resulted in significant "hits." Taken together, these data indicate that the presence or absence of Hbr elements is a significant contributory factor to the high level of polymorphism associated with maize genic regions.

Base Sequence↗
PubMed2000

The rice R gene family: two distinct subfamilies containing several miniature inverted-repeat transposable elements.

The R and B genes of maize regulate the anthocyanin biosynthetic pathway and constitute a small gene family whose evolution has been shaped by polyploidization and transposable element activity. To compare the evolution of regulatory genes in the distinct but related genomes of rice and maize, we previously isolated two R homologues from rice (Oryza sativa). The Ra1 gene on chromosome 4 can activate the anthocyanin pathway, whereas the Rb gene, of undetermined function, maps to chromosome 1. In this study, rice R genes have been further characterized. First, we found that an Rb cDNA can induce pigmentation in maize suspension cells. Second, another rice R homologue (Ra2) was identified that is more closely related to Ra1 than to Rb. Domesticated rice and its wild relatives harbor multiple Ra-like and Rb-like genes despite the fact that rice is a true diploid with the smallest genome of all the grass species analyzed to date. Finally, several miniature inverted-repeat transposable elements (MITEs) were found in R family members. Their possible role in hastening the divergence of R genes is discussed.

Amino Acid Sequence↗
PubMed2000

Antisense waxy genes with highly active promoters effectively suppress waxy gene expression in transgenic rice.

To regulate Waxy (Wx) gene expression by introducing antisense genes, we connected the 2.3 kb Wx cDNA having 450 bp of the Wx first intron in reverse orientation to rice Wx and maize alcohol dehydrogenase1 (Adh1) promoters and used these constructs to transform rice plants. Of 10 independent transgenic lines analysed, four lines showed various degrees of reduction in amylose and WAXY (WX) protein levels in the endosperm. In two transgenic lines, complete absence of amylose was observed which made the seeds opaque white like glutinous rice (amylose-deficient waxy (wx) mutant). In one of the transgenic lines, A1 line, the presence of the antisense Wx gene cosegregated with reduction of amylose content in the endosperm. In the same line, a reduction in the level of endogenous Wx mRNA was observed in immature endosperm. Interestingly, this reduction was observed only with mature spliced transcripts but not with unspliced transcripts. Reduced amylose synthesis was also observed in pollen grains of four transgenic lines. These results suggest that integrated antisense Wx gene caused a reduction in amylose synthesis in endosperms and pollen grains of transgenic rice carrying the antisense Wx cDNA. These results indicate that manipulation of starch and other carbohydrates in rice grain is possible using antisense genes.

Amylose↗
PubMed1998

A naturally occurring functional allele of the rice waxy locus has a GT to TT mutation at the 5' splice site of the first intron.

In cultivated rice two wild-type alleles, Wxa and Wxb, predominate at the waxy locus, which encodes granule-bound starch synthase. The activity of Wxa is 10-fold higher than that of Wxb at the level of both protein and mRNA. Wxb has a +1G to T mutation at the 5' splice site of the first intron. Sequence analysis of Wxb transcripts revealed that splicing occurs at the mutant AG/UU site and at two cryptic sites: the first is A/GUU, one base upstream of the original site and the second is AG/GU found approximately 100 bases upstream of the mutant splice site. We introduced single base mutations to the 5' splice sites of both Wxa and Wxb, fused with the gus reporter gene and introduced them into rice protoplasts. Analysis of GUS activities and transcripts indicated that a G to T mutation in Wxa reduced GUS activity and the level of spliced RNA. Conversely, a T to G mutation of Wxb restored GUS activity and the level of spliced RNA to that of wild-type Wxa. These results demonstrated that the low level expression of Wxb results from a single base mutation at the 5' splice site of the first intron. It is of interest that the Wxb allele of rice carrying the G to T mutation of intron 1 has been conserved in the history of rice cultivation because there is a low amylose content of the seed caused by this mutation.

Alleles↗
PubMed1998

Extreme structural heterogeneity among the members of a maize retrotransposon family.

A few families of retrotransposons characterized by the presence of long terminal repeats (LTRs) have amplified relatively recently in maize and account for >50% of the genome. Surprisingly, none of these elements have been shown to cause a single mutation. In contrast, most of the retrotransposon-induced mutations isolated in maize are caused by the insertion of elements that are present in the genome at 2-50 copies. To begin to understand what limits the amplification of this mutagenic class of LTR-retrotransposons, we are focusing on five elements previously identified among 17 mutations of the maize waxy gene. One of these elements, Stonor, has sustained a deletion of the entire gag region and part of the protease domain. Missing sequences were recovered from larger members of the Stonor family and indicate that the deletion probably occurred during retrotransposition. These large elements have an exceptionally long leader of 2 kb that includes a highly variable region of approximately 1 kb that has not been seen in previously characterized retrotransposons. This region serves to distinguish each member of the Stonor family and indicates that no single element has yet evolved that can attain the very high copy numbers characteristic of other element families in maize.

Amino Acid Sequence↗
PubMed1998

Molecular consequences of Ds insertion into and excision from the helix-loop-helix domain of the maize R gene.

The R and B proteins of maize are required to activate the transcription of several genes in the anthocyanin biosynthetic pathway. To determine the structural requirements for R function in vivo, we are exploiting its sensitive mutant phenotype to identify transposon (Ds) insertions that disrupt critical domains. Here we report that the ability of the r-m1 allele to activate transcription of at least three structural genes is reduced to only 2% of wild-type activity because of a 396-bp Ds element in helix 2 of the basic helix-loop-helix (bHLH) motif. Residual activity likely results from the synthesis of a mutant protein that contains seven additional amino acids in helix 2. This protein is encoded by a transcript where most of the Ds sequence has been spliced from pre-mRNA. Two phenotypic classes of stable derivative alleles, very pale and extremely pale, condition <1% of wild-type activity as a result of the presence of two- and three-amino-acid insertions, respectively, at the site of Ds excision. Localization of these mutant proteins to the nucleus indicates a requirement for an intact bHLH domain after nuclear import. The fact that deletion of the entire bHLH domain has only a minor effect on R protein activity while these small insertions virtually abolish activity suggests that deletion of the bHLH domain may bypass a requirement for bHLH-mediated protein-protein interactions in the activation of the structural genes in the anthocyanin biosynthetic pathway.

Alleles↗
PubMed1998

Inefficient reinitiation is responsible for upstream open reading frame-mediated translational repression of the maize R gene.

Maize R genes encode a small family of transcriptional activators of several structural genes in the anthocyanin biosynthetic pathway. The 5' leader region of most R genes contains a 38-codon upstream open reading frame (uORF) that previously was shown to be responsible for the repression of downstream gene expression in a transient transformation assay. In this study, we report that the 5' leader also can repress translation of the downstream luciferase gene both in the rabbit reticulocyte translation system and in transgenic rice plants. The ability to visualize the uORF peptide after in vitro translation permits quantification of both products of dicistronic mRNAs. Similarly, the construction of transgenic rice plants expressing wild-type and mutant constructs permits the quantification and correlation of steady state mRNA levels and reporter gene activities. Using these assays, we demonstrate directly that translation of the uORF is required for repression, that increasing translation of the uORF peptide decreases downstream gene expression, and that repression is unaffected by either subtle or gross changes in the uORF peptide. Rather, we find that ribosomes that translate the uORF reinitiate inefficiently and that the intercistronic sequence downstream of the uORF mediates this effect.

Amino Acid Sequence↗
PubMed1998

Transposable elements and the evolution of gene expression.

Most plant genomes are populated with enormous quantities of transposable elements (TEs) or sequences derived from TEs. The impact of TEs on their host has been addressed by characterizing mutations of the maize waxy and R genes caused by TE insertions. Association between a new class of TEs (called MITEs) and normal plant genes is also reviewed. The notion that different classes of TEs have found their respective niches in the maize genome is discussed.

DNA Transposable Elements↗
PubMed1997

Retrotransposon insertion into the maize waxy gene results in tissue-specific RNA processing.

We previously reported that three alleles of the maize waxy (wx) gene were alternatively spliced as a result of the insertion of retrotransposons into intronic sequences. In addition, inefficient splicing of element sequences with the surrounding intron produced wild-type transcripts that presumably were responsible for the observed residual gene expression in the endosperm. In this study, we report that one of these alleles, wxG, has a tissue-specific phenotype with 30-fold more WX enzymatic activity in pollen than in the endosperm. Quantification of wxG-encoded transcripts in pollen and the endosperm demonstrates that this difference can be accounted for by tissue-specific differences in RNA processing. Specifically, there is approximately 30-fold more correctly spliced RNA in pollen than in the endosperm. Based on an analogy to similar examples of tissue-specific alternative splicing in animal systems, we hypothesize that the tissue-specific phenotype of the wxG allele may reflect differences in the concentration of splicing factors in these tissues.

Alleles↗
PubMed1996

A computer-based systematic survey reveals the predominance of small inverted-repeat elements in wild-type rice genes.

Several recent reports indicate that mobile elements are frequently found in and flanking many wild-type plant genes. To determine the extent of this association, we performed computer-based systematic searches to identify mobile elements in the genes of two "model" plants, Oryza sativa (domesticated rice) and Arabidopsis thaliana. Whereas 32 common sequences belonging to nine putative mobile element families were found in the noncoding regions of rice genes, none were found in Arabidopsis genes. Five of the nine families (Gaijin, Castaway, Ditto, Wanderer, and Explorer) are first described in this report, while the other four were described previously (Tourist, Stowaway, p-SINE1, and Amy/LTP). Sequence similarity, structural similarity, and documentation of past mobility strongly suggests that many of the rice common sequences are bona fide mobile elements. Members of four of the new rice mobile element families are similar in some respects to members of the previously identified inverted-repeat element families, Tourist and Stowaway. Together these elements are the most prevalent type of transposons found in the rice genes surveyed and form a unique collection of inverted-repeat transposons we refer to as miniature inverted-repeat transposable elements or MITEs. The sequence and structure of MITEs are clearly distinct from short or long interspersed nuclear elements (SINEs or LINEs), the most common transposable elements associated with mammalian nuclear genes. Mobile elements, therefore, are associated with both animal and plant genes, but the identity of these elements is strikingly different.

Arabidopsis↗