Richard L. Converse

Role of Lumican in the Corneal Epithelium during Wound Healing

Lumican regulates collagenous matrix assembly as a keratan sulfate proteoglycan in the cornea and is also present in the connective tissues of other organs and embryonic corneal stroma as a glycoprotein. In normal unwounded cornea, lumican is expressed by stromal keratocytes. Our data show that injured mouse corneal epithelium ectopically and transiently expresses lumican during the early phase of wound healing, suggesting a potential lumican functionality unrelated to regulation of collagen fibrillogenesis,e.g. modulation of epithelial cell adhesion or migration. An anti-lumican antibody was found to retard corneal epithelial wound healing in cultured mouse eyes. Healing of a corneal epithelial injury in Lum −/− mice was significantly delayed compared with Lum +/− mice. These observations indicate that lumican expressed in injured epithelium may modulate cell behavior such as adhesion or migration, thus contributing to corneal epithelial wound healing.

Cornea-specific expression of K12 keratin during mouse development

The full-length cDNA of mouse K12 keratin was characterized by sequencing overlapping cDNA clones isolated from a mouse cornea cDNA library. Using Northern blot hybridization, the radio-labeled cDNA hybridized to a 1.9 kb mRNA from adult cornea, but not from other mouse tissues including snout, esophagus, tongue, and skin. During mouse development, corneas do not express K12 mRNA until 4 days postnatal when the epithelium begins to stratify as judged by Northern blot and in situ hybridization. In situ hybridization with 3H-labeled cDNA probe and immunohistochemical studies with antibodies against a synthetic oligo-peptide deduced from rabbit K12 cDNA demonstrate that this mouse K12 keratin is expressed in all cell layers of adult corneal epithelium, and the suprabasal layers, but not the basal layer of the limbal epithelium. Epidermal growth factor (EGF) has been shown to promote epithelium stratification of cultured chicken and human corneas in vitro. To examine whether EGF can promote K12 expression, EGF was administered to neonatal mice. The results indicate that EGF retards K12 expression by corneal epithelial cells, even though it promotes corneal epithelial stratification during mouse development. Taken together, our results demonstrate that the expression of K12 keratin is cornea-specific, differentiation-dependent, and developmentally regulated.

CHARACTERIZATION AND EXPRESSION OF THE MOUSE LUMICAN GENE

Lumican is one of the major keratan sulfate proteoglycans (KSPG) in vertebrate corneas. We previously cloned the murine lumican cDNA. This study determines the structure of murine lumican gene (Lum) and its expression during mouse embryonic developments. The mouse lumican gene was isolated from a bacterial artificial chromosome mouse genomic DNA library and characterized by polymerase chain reaction and Southern hybridization. The lumican gene spans 6.9 kilobase pairs of mouse genome. The gene consists of three exons and two introns. Exon 1 constitutes 88 bases (b) of untranslated sequence. Exon 2 is 883 b and contains most of the coding sequence of lumican mRNA, and exon 3 has 152 b of coding sequence and 659 b of 3′ noncoding sequence. The mouse lumican gene has a TATCA element, a presumptive TATA box, which locates 27 b 5′-upstream from the transcription initiation site. Northern hybridization and in situ hybridization indicate that in early stages of embryonic development, day 7 post coitus the embryo expresses little or no lumican. Thereafter, different levels of lumican mRNA can be detected in various organ systems, such as cornea stroma, dermis, cartilage, heart, lung, and kidney. The cornea and heart are the two tissues that have the highest expression in adult. Immunoblotting studies found that KSPG core proteins became abundant in the cornea and sclera by postnatal day 10 but that sulfated KSPG could not be detected until after the eyes open. These results indicate that lumican is widely distributed in most interstitial connective tissues. The modification of lumican with keratan sulfates in cornea is concurrent with eye opening and may contribute to corneal transparency.

The Cloning of Mouse Keratocan cDNA and Genomic DNA and the Characterization of Its Expression during Eye Development

Keratan sulfate proteoglycans (KSPGs) play a pivotal role in the development and maintenance of corneal transparency. Keratocan, lumican, and mimecan (osteoglycin) are the major KSPGs in vertebrate corneas. To provide a better understanding of the structure/function relationship of keratocan, we have cloned both the mouse keratocan gene and its cDNA. We have also examined its expression during embryonic development. The mouse keratocan gene spans approximately 6.5 kilobases of the mouse genome and contains three exons and two introns. Northern blotting and in situhybridization were employed to examine keratocan gene expression during mouse development. Unlike lumican gene, which is expressed by many tissues other than cornea, keratocan mRNA is more selectively expressed in the corneal tissue of the adult mouse. During embryonic development, keratocan mRNA was first detected in periocular mesenchymal cells migrating toward developing corneas on embryonic day 13.5 (E13.5). Its expression was gradually restricted to corneal stromal cells on E14.5∼E18.5. Interestingly, keratocan mRNA can be detected in scleral cells of E15.5 embryos, but not in E18.5 embryos. In adult eyes, keratocan mRNA can be detected in corneal keratocytes, but not in scleral cells.

QTL analysis for early-maturing traits in cotton using two upland cotton (Gossypium hirsutum L.) crosses

Making use of the markers linked closely to QTL for early-maturing traits for MAS (Marker-assisted selection) is an effective method for the simultaneous improvement of early maturity and other properties in cotton. In this study, two F2 populations and their F2:3 families were generated from the two upland cotton (Gossypium hirsutum L.) crosses, Baimian2 × TM-1 and Baimian2 × CIR12. QTL for early-maturing traits were analyzed using F2:3 families. A total of 54 QTL (31 suggestive and 23 significant) were detected. Fourteen significant QTL had the LOD scores not only > 3 but also exceeding permutation threshold. At least four common QTL, qBP-17 for bud period (BP), qGP-17a/qGP-17b (qGP-17) for growth period (GP), qYPBF-17a/qYPBF-17b (qYPBF-17) for yield percentage before frost (YPBF) and qHFFBN-17 for height of first fruiting branch node (HFFBN), were found in both populations. These common QTL should be reliable and could be used for MAS to facilitate early maturity. The common QTL, qBP-17, had a LOD score not only > 3 but also exceeding permutation threshold, explaining 12.6% of the phenotypic variation. This QTL should be considered preferentially in MAS. Early-maturing traits of cotton are primarily controlled by dominant and over-dominant effects.

Molecular Analysis and Characterization of Zebrafish Keratocan (zKera) Gene

Corneal small leucine-rich proteoglycans play a pivotal role in maintaining corneal transparency and function. In this study, we isolated and characterized the zebrafish (Danio rerio) keratocan (zKera) gene. The human keratocan sequence was used to search zebrafish homologues. The zKera full-length genomic DNA and cDNA were generated via PCR of zebrafish genomic DNA and reverse transcription-PCR of total zebrafish eye RNA, respectively. The zKera spanning 3.5 kilobase pairs consists of two exons and one intron and a TATA-less promoter. The zKera encodes 341 amino acids with 59% identity to its human counterpart and 57% identity to that of mouse keratocan. Like mouse and chick keratocan, zKera mRNA is selectively expressed in the adult cornea; however, during embryonic development, zKera mRNA is expressed in both the brain and the cornea. Interestingly, it is expressed mainly in corneal epithelium but also in the stroma. A pseudogene was proved by introducing a zKera promoter-driven enhanced green fluorescence protein reporter gene into fertilized zebrafish eggs. Using morpholino-antisense against zKera to knock down zKera resulted in a lethal phenotype due to massive caspase-dependent apoptosis, which was noted by a significant increase of active caspase-3 and caspase-8 in the developing forebrain area, including the eyes. This is different from mouse, for which keratocan-deficient mice are viable. Taken together, our data indicate that mammalian keratocan is conserved in zebrafish in terms of gene structure, expression pattern, and promoter function.

Identification of Interacting Motifs Between Armadillo Repeat Containing 1 (ARC1) and Exocyst 70 A1 (Exo70A1) Proteins in Brassica oleracea

In order to identify the functional domains which regulate the interaction between the self-incompatibility proteins armadillo repeat containing 1 (ARC1) and exocyst 70 A1 (Exo70A1) in Brassica oleracea, fragments containing selected motifs of ARC1 (ARC1210, ARC1246, ARC1279, ARC1354) and site-specific mutants with substitutions at possible interaction sites (ARC1354m, ARC1664m) were PCR amplified and inserted into pGADT7, while coding sequences from Exo70A1 (Exo70A185, Exo70A1) were subcloned into pGBKT7. The interactions between the protein products produced by these constructs were then analyzed utilizing a yeast two-hybrid system. Our data indicate that both ARC1210 and ARC1246 interact strongly with Exo70A185 and Exo70A1, while ARC1279, ARC1354, ARC1354m and ARC1664m exhibited a weak interaction, indicating that the recognition sites are located within the 210 N-terminal amino acids of ARC1 and the 85 N-terminal amino acids of Exo70A1. This was further verified by GST pull-down analysis. This supports a model in which the N-terminal leucine zipper of ARC1 and the first 85 N-terminal amino acids of Exo70A1 mediate the interaction between these two proteins. Bioinformatic and phylogenetic analysis demonstrated that these motifs were highly conserved across different species, indicating that the interaction characterized in B. oleracea may operate in a wide array of cultivars.

Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences

The compactness of plant chromosomes and the structure of the plant cell wall and cytoplasm provide a great obstacle to fluorescence in situ hybridization (FISH) for single-copy or low-copy DNA sequences. Consequently, many new methods for improving spatial resolution via chromosomal stretching have been employed to overcome this technical challenge. In this article, a technique for extracting cell-wall free nuclei at mitotic interphase, then using these nuclei to prepare extended DNA fibers (EDFs) by the method of a receding interface, whereby slide-mounted chromatin produces EDFs in concert with gravity-assisted buffer flow, was adopted as a result of the low frequency of EDF damage produced by this procedure. To examine the quality of these EDFs, we used single-copy gene encoding S-locus receptor kinase and multi-copy 5S rDNA (ribosomal DNA) as probes. The resulting EDFs proved suitable for high-resolution FISH mapping for repetitive DNA sequences, and the localization of a single-copy locus.

Interaction Between Two Self-Incompatible Signal Elements, EXO70A1 and ARC1

Abstract ARC1 and EXO70A1 are important signal elements of self-incompatibility in Brassica . To characterize the interaction of ARC1-EXO70A1 during the course of self-incompatibility, the coding sequences of ARC1 and EXO70A1 were cloned from Brassica napus L. and B. oleracea L. var. acephala . Sequence analysis showed that ARC1 consisted of 663 amino acids in B. oleracea and 661 amino acids in B. napus , with a 45-amino-acid difference between them. Sequence alignment showed 95.9% similarity, with 93.9% exact match between BoARC1 and BnARC1. Only a 6-amino-acid difference was detected between BoEXO70A1 and BnEXO70A1, with 99.4% similarity and 98.9% exact match according to further sequence alignment. The homology between EXO70A1 alleles was higher than that between ARC1 alleles. Yeast 2-hybrid results indicated that a strong interaction existed between ARC1 and EXO70A1, which could activate the expressions of 4 reporter genes ( ADE2 , HIS3 , AUR1-C , and MEL1 ) in diploid yeast. However, there was very weak interaction between EXO70A1 and a 316-C-terminal-deletion mutant of ARC1 (ARC1 N ), which only activated the expressions from 3 reporter genes ( ADE2 , AUR1-C , and MEL1 ). This indicated that the interaction interface between ARC1 and EXO70A1 might not reside within the Armadillo (ARM) repeat domains of ARC1. The N-terminal domains of ARC1 play an essential role in the interaction of ARC1 with EXO70A1. The influence of the differences in amino-acid composition between BoARC1 and BnARC1 on the interaction between ARC1-EXO70A1 was not detected with a yeast 2-hybrid system, which may indicate that the binding interface between ARC1 and EXO70A1 was not altered by sequence differences between the 2 proteins in these Brassica species.

A novel dominant rice male sterility mutant, OsDMS-1, simultaneously controlled by independent loci on chromosomes 1, 2, and 3

We found a rice dominant genetic male-sterile mutant OsDMS-1 from tissue culture-regenerated offspring of Zhonghua 11 (japonica rice). Compared to Zhonghua 11, OsDMS-1 mutant anthers were narrow and pale and incapable of pollen release although the glume opened normally. Approximately 81.4% of this mutant pollen was small and malformed and could not be stained by iodine treatment. A paraffin section assay showed delayed degradation of the OsDMS-1 mutant tapetum without starch accumulation in the mutant pollen, ultimately leading to pollen abortion. Classical genetic analysis indicated that only one dominant gene controlled the sterility in the OsDMS-1 mutant. However, molecular mapping suggested three loci simultaneously control male sterility in this mutant: OsDMS-1A (on chromosome 1), flanked by InDel markers C1D4 and C1D5, OsDMS-1B (on chromosome 2), flanked by InDel markers C2D3 and C2D10, and OsDMS-1C (on chromosome 3), flanked by InDel markers 0315 and C3D3. Molecular mapping disagreed with classical genetic analysis regarding the number of genes controlling the OsDMS-1 mutant, indicating a novel mechanism underlying sterility in OsDMS-1. We present two hypotheses to explain this novel inheritance behavior: one is described as Parent-Originated Loci Tying Inheritance (POLTI); while the alternate hypothesis is described as Loci Recombination Lethal (LRL).