Biomedical subjects

Michael Freeling

Publications and source records attributed to Michael Freeling.

11 recordsLinked to original sources

PubMed2004

Regulation and a conserved intron sequence of liguleless3/4 knox class-I homeobox genes in grasses.

The nine class-I maize (Zea mays L.) knox genes are putative transcription factors normally expressed in shoot apices, but not in leaves. knotted1 (kn1) seems to function in shoot apical meristem maintenance, and rough sheath1 (rs1)-like genes may act in internode elongation. The function of liguleless3 (lg3)-type genes is still unknown. Here, we characterized lg3 as well as the two most closely related genes liguleless4a (lg4a, formerly knox11) and liguleless4b (lg4b, formerly knox5). We termed this subclass of knox genes lg3/4 genes. We studied the expression patterns of lg3/4 genes and compared their sequences. We obtained knockout mutants of lg3 by finding Mu transposon insertions into exons. Our results show that lg3 was not essential for plant development, and that lg4a and lg4b were likely to encode the redundant function. In addition, lg4a but not lg4b was ectopically expressed in the Lg4-O mutant, suggesting that this mutant was affected at the lg4a locus. We found that the lg3 gene was unique among knox genes as it was co-induced in the leaves of leaf mutants that ectopically expressed knox genes in the leaves. The leaf phenotype expressed in the dominant Rs1-O mutant was not altered when lg3 function was removed using the knockout. Genomic sequence comparisons of lg3, lg4a and lg4b from maize and the two homologous genes, osh6 and osh71, from rice revealed a 14-bp phylogenetic footprint in intron II. This sequence was conserved in nucleotide composition, position and polarity in the lg3/4 genes of divergent grasses representing six Gramineae subfamilies. In an independent experiment, this same conserved sequence was found in a yeast reverse one-hybrid screen for putative binding sites of the LG3 homeodomain protein. Distribution of this 14-bp sequence was examined within the public rice database. The possible function of this sequence in regulation of lg3/4 genes is discussed.

Amino Acid Sequence↗

PubMed2004

Genomic duplication, fractionation and the origin of regulatory novelty.

Having diverged 50 MYA, rice remained diploid while the maize lineage became tetraploid and then fractionated by losing genes from one or the other duplicate region. We sequenced and annotated 13 maize genes (counting the duplicate gene as one gene) on one or the other of the pair of homeologous maize regions; 12 genes were present in one cluster in rice. Excellent maize-rice synteny was evident, but only after the fractionated maize regions were condensed onto a finished rice map. Excluding the gene we used to define homeologs, we found zero retention. Once retained, fractionation (loss of functioning DNA sequence) could occur within cis-acting gene space. We chose a retained duplicate basic leucine zipper transcription factor gene because it was well marked with big, exact phylogenetic footprints (CNSs). Detailed alignments of lg2 and retained duplicate lrs1 to their rice ortholog found that fractionation of conserved noncoding sequences (CNSs) was rare, as expected. Of 30 CNSs, 27 were conserved. The 3 unexpected, missing CNSs and a large insertion support subfunctionalization as a reflection of fractionation of cis-acting gene space and the recent evolution of lg2's novel maize leaf and shoot developmental functions. In general, the principles of fractionation and consolidation work well in making sense of maize gene and genomic sequence data.

Chromosomes, Artificial, Bacterial↗

PubMed2003

Conservation and molecular dissection of ROUGH SHEATH2 and ASYMMETRIC LEAVES1 function in leaf development.

Maize ROUGH SHEATH2 (RS2) and Arabidopsis ASYMMETRIC LEAVES1 (AS1) are orthologous Myb-related genes required for leaf development and act as negative regulators of class 1 KNOTTED1-like homeobox (KNOX) genes in leaf primordia. Expression of RS2 in Arabidopsis fully complements as1 leaf phenotypes and represses the expression of the KNOX gene KNAT1 in leaves. Whereas loss of AS1 function in Arabidopsis results in rounded, lobed leaves with shorter and wider petioles, overexpression of either RS2 or AS1 results in longer and narrower leaves with longer petioles than wild type. A conserved C-terminal domain (CTD) mediates homodimerization of both RS2 and AS1 and modulates leaf shape when expressed independently of the Myb domain in Arabidopsis. Homodimerization is not absolutely required for KNAT1 repression. RS2:GFP fusion protein is biologically active, localized in discrete dynamic subnuclear foci and associates with DNA during cell division.

Arabidopsis↗

PubMed2003

ZmDB, an integrated database for maize genome research.

Zea mays DataBase (ZmDB) seeks to provide a comprehensive view of maize (corn) genetics by linking genomic sequence data with gene expression analysis and phenotypes of mutant plants. ZmDB originated in 1999 as the Web portal for a large project of maize gene discovery, sequencing and phenotypic analysis using a transposon tagging strategy and expressed sequence tag (EST) sequencing. Recently, ZmDB has broadened its scope to include all public maize ESTs, genome survey sequences (GSSs), and protein sequences. More than 170 000 ESTs are currently clustered into approximately 20 000 contigs and about an equal number of apparent singlets. These clusters are continuously updated and annotated with respect to potential encoded protein products. More than 100 000 GSSs are similarly assembled and annotated by spliced alignment with EST and protein sequences. The ZmDB interface provides quick access to analytical tools for further sequence analysis. Every sequence record is linked to several display options and similarity search tools, including services for multiple sequence alignment, protein domain determination and spliced alignment. Furthermore, ZmDB provides web-based ordering of materials generated in the project, including ESTs, ordered collections of genomic sequences tagged with the RescueMu transposon and microarrays of amplified ESTs. ZmDB can be accessed at http://zmdb.iastate.edu/.

DNA Transposable Elements↗

PubMed2003

Mu killer causes the heritable inactivation of the Mutator family of transposable elements in Zea mays.

Mutations in a number of genes responsible for the maintenance of transposon silencing have been reported. However, the initiation of epigenetic silencing of transposable elements is poorly characterized. Here, we report the identification of a single dominant locus, Mu killer (Muk), that acts to silence MuDR, the autonomous regulatory transposon of the Mutator family of transposable elements in maize. Muk results in the methylation of MuDR TIRs and is competent to silence one or several active MuDR elements. Silencing by Muk is not dependent on the position of the MuDR element and occurs gradually during plant development. Transcript levels of the MuDR transposase, mudrA, decrease substantially when Muk is present. The other transcript encoded by MuDR, mudrB, also fails to accumulate in the poly(A) RNA fraction when MuDR and Muk are combined. Additionally, plants undergoing MuDR silencing produce small, mudrA-homologous approximately 26-nt RNAs, suggesting a role for RNA-directed DNA methylation in MuDR silencing. MuDR elements silenced by Muk remain silenced even in plants that do not inherit Muk, suggesting that Muk is required for the initiation of MuDR silencing but not for its maintenance.

DNA Methylation↗

PubMed2003

The extended auricle1 (eta1) gene is essential for the genetic network controlling postinitiation maize leaf development.

The maize leaf is composed of distinct regions with clear morphological boundaries. The ligule and auricle mark the boundary between distal blade and proximal sheath and are amenable to genetic study due to the array of mutants that affect their formation without severely affecting viability. Herein, we describe the novel maize gene extended auricle1 (eta1), which is essential for proper formation of the blade/sheath boundary. Homozygous eta1 individuals have a wavy overgrowth of auricle tissue and the blade/sheath boundary is diffuse. Double-mutant combinations of eta1 with genes in the knox and liguleless pathways result in synergistic and, in some cases, dosage-dependent interactions. While the phenotype of eta1 mutant individuals resembles that of dominant knox overexpression phenotypes, eta1 mutant leaves do not ectopically express knox genes. In addition, eta1 interacts synergistically with lg1 and lg2, but does not directly affect the transcription of either gene in leaf primordia. We present evidence based on genetic and molecular analyses that eta1 provides a downstream link between the knox and liguleless pathways.

Base Sequence↗

PubMed2003

Conserved noncoding sequences in the grasses.

As orthologous genes from related species diverge over time, some sequences are conserved in noncoding regions. In mammals, large phylogenetic footprints, or conserved noncoding sequences (CNSs), are known to be common features of genes. Here we present the first large-scale analysis of plant genes for CNSs. We used maize and rice, maximally diverged members of the grass family of monocots. Using a local sequence alignment set to deliver only significant alignments, we found one or more CNSs in the noncoding regions of the majority of genes studied. Grass genes have dramatically fewer and much smaller CNSs than mammalian genes. Twenty-seven percent of grass gene comparisons revealed no CNSs. Genes functioning in upstream regulatory roles, such as transcription factors, are greatly enriched for CNSs relative to genes encoding enzymes or structural proteins. Further, we show that a CNS cluster in an intron of the knotted1 homeobox gene serves as a site of negative regulation. We showthat CNSs in the adh1 gene do not correlate with known cis-acting sites. We discuss the potential meanings of CNSs and their value as analytical tools and evolutionary characters. We advance the idea that many CNSs function to lock-in gene regulatory decisions.

5' Flanking Region↗

PubMed2003

Combinatorial control of meristem identity in maize inflorescences.

The architecture of maize inflorescences, the male tassel and the female ear, is defined by a series of reiterative branching events. The inflorescence meristem initiates spikelet pair meristems. These in turn initiate spikelet meristems which finally produce the floret meristems. After initiating one meristem, the spikelet pair and spikelet meristem convert into spikelet and floret meristems, respectively. The phenotype of reversed germ orientation1 (rgo1) mutants is the production of an increased number of floret meristems by each spikelet meristem. The visible phenotypes include increased numbers of flowers in tassel and ear spikelets, disrupted rowing in the ear, fused kernels, and kernels with embryos facing the base of the ear, the opposite orientation observed in wild-type ears. rgo1 behaves as single recessive mutant. indeterminate spikelet1 (ids1) is an unlinked recessive mutant that has a similar phenotype to rgo1. Plants heterozygous for both rgo1 and ids1 exhibit nonallelic noncomplementation; these mutants fail to complement each other. Plants homozygous for both mutations have more severe phenotypes than either of the single mutants; the progression of meristem identities is retarded and sometimes even reversed. In addition, in rgo1; ids1 double mutants extra branching is observed in spikelet pair meristems, a meristem that is not affected by mutants of either gene individually. These data suggest a model for control of meristem identity and determinacy in which the progress through meristem identities is mediated by a dosage-sensitive pathway. This pathway is combinatorially controlled by at least two genes that have overlapping functions.

Crosses, Genetic↗

PubMed2002

Utility and distribution of conserved noncoding sequences in the grasses.

Control of gene expression requires cis-acting regulatory DNA sequences. Historically these sequences have been difficult to identify. Conserved noncoding sequences (CNSs) have recently been identified in mammalian genes through cross-species genomic DNA comparisons, and some have been shown to be regulatory sequences. Using sequence alignment algorithms, we compared genomic noncoding DNA sequences of the liguleless1 (lg1) genes in two grasses, maize and rice, and found several CNSs in lg1. These CNSs are present in multiple grass species that represent phylogenetically disparate lineages. Six other maize/rice genes were compared and five contained CNSs. Based on nucleotide substitution rates, these CNSs exist because they have biological functions. Our analysis suggests that grass CNSs are smaller and far less frequent than those identified in mammalian genes and that mammalian gene regulation may be more complex than that of grasses. CNSs make excellent pan-grass PCR-based genetic mapping tools. They should be useful as characters in phylogenetic studies and as monitors of gene regulatory complexity.

Animals↗

PubMed2002

Biodiversity (Communications arising): maize transgene results in Mexico are artefacts.

Quist and Chapela's conclusion that the transgenes they claim to have detected in native maize in Oaxaca, Mexico, are predominantly reassorted and inserted into a "diversity of genomic contexts" seems to be based on an artefact arising from the inverse polymerase chain reaction (i-PCR) they used to amplify sequences flanking 35S transgenes from cauliflower mosaic virus (CaMV).

Artifacts↗

PubMed2002

Expression of a mutant maize gene in the ventral leaf epidermis is sufficient to signal a switch of the leaf's dorsoventral axis.

Maize leaves are initiated from the shoot apex with an inherent leaf dorsoventral polarity; the leaf surface closest to the meristem is the adaxial (upper, dorsal) surface whereas the opposite leaf surface is the abaxial (lower, ventral) surface. The Rolled leaf1 (Rld1) semi-dominant maize mutations affect dorsoventral patterning by causing adaxialization of abaxial leaf regions. This adaxialization is sometimes associated with abaxialization of the adaxial leaf regions, which constitutes a "switch". Dosage analysis indicates Rld1 mutants are antimorphs. We mapped Rld1's action to a single cell layer using a mosaic analysis and show Rld1 acts non cell-autonomously along the dorsoventral axis. The presence of Rld1 mutant product in the abaxial epidermis is necessary and sufficient to induce the Rolled leaf1 phenotype within the lower epidermis as well as in other leaf layers along the dorsoventral axis. These results support a model for the involvement of wild-type RLD1 in the maintenance of dorsoventral features of the leaf. In addition, they demonstrate the abaxial epidermis sends/receives a cell fate determining signal to/from the adaxial epidermis and controls the dorsoventral patterning of the maize leaf.

Gene Expression↗