Supplementary_table – Supplemental material for Genetic variants in miRNA machinery genes associated with clinicopathological characteristics and outcomes of gastric cancer patients

<p>Supplemental material, Supplementary_table for Genetic variants in miRNA machinery genes associated with clinicopathological characteristics and outcomes of gastric cancer patients by Yuqian Liao, Yulu Liao, Jun Li, Liyan Liu, Junyu Li, Yiye Wan and Lixiang Peng in The International Journal of Biological Markers</p

RT-PCR analysis of <i>OsCESA7</i> expression.

<p>(A) Semiquantitative RT-PCR analysis of <i>OsCESA9</i> expression. (B) Real-time RT-PCR analysis of <i>OsCESA7</i> expression. Total RNA was extracted from leaf sheaths, leaf blades, and roots at the seedling stage and from culms and panicles at the mature stage of wild-type plants. The rice <i>ACTIN1</i> gene was used as a control for equal loading. Values shown are averages of four replicates. Bars represent standard errors.</p

Map-based cloning of the gene responsible for the S1-24 phenotype.

<p>(A) The location of the gene locus was narrowed to an approximately 154-kb region on chromosome 10. Vertical lines represent the positions of molecular markers and the number of recombinants. (B) Seventeen predicted ORFs within the fine mapping region. Green, ORFs with known biochemical functions; Yellow, ORFs encoding expressed hypothetical proteins; Black, ORFs encoding transposons. (C) Genomic structure of <i>OsCESA7</i>. Boxes indicate exons. The mutation site is located in the first exon. (D) Protein structure of OsCESA7 including the RING-type zinc finger indicated in blue; two Asp (D) residues, the DXD, Q/RXXRW motifs indicated in red; and eight transmembrane domains indicated in yellow. Tos17 insertion sites in the NC0259 and ND8759 mutants allelic to the S1-24 mutant are indicated by arrows. (E) Alignment of zinc finger motif template and the corresponding OsCESA7 region. The site of the mutation C40 is highly conserved.</p

Phenotypes and physical properties of the S1-24 mutant.

<p>(A) An easily broken culm of S1-24 compared with the wild type. (B) An easily broken flag leaf of S1-24 compared with the wild type. (C, D) Force required to break the first and second upper internodes. (E, F) Elongation length of the first and second upper internodes. Values shown are the averages of values for five internodes. Bars represent standard errors. ** indicate statistical significance by a <i>t</i> test at <i>P</i> < 0.01.</p

Expression pattern of OsCESA7 revealed by GUS-staining in OsCESA7promoter: GUS transgenic plants.

<p>(A) A segment of leaf blade. (B) Leaf blade cross section. (C) Magnified image of the root. (D) Leaf sheath. (E) Leaf sheath cross section. (F) Spikelet. (G) Stem. (H) Stem cross section. Signals were detected in vascular bundles, especially in sclerenchyma cells.</p

Phenotypes of genetic complementation in transgenic S1-24 mutant plants.

<p>(A) Gross morphologies of wild-type, S1-24, and R1 and R2 complementation lines at the mature stage. (B) Resistance to breakage in culms of the R1 and R2 complementation lines compared with S1-24 and wild-type culms.</p

Cross section of a culm viewed under a scanning electron microscope.

<p>(A, B) Cross section of a wild-type culm. (C, D) Cross section of an S1-24 culm. Magnification in the images is 500× (A, C) or 5000 × (B, D). Green and yellow arrows represented thickened sclerenchyma cell (SC) walls and unthicken parenchyma cell (PC) walls.</p

The site of the mutation in the S1-24 mutant and phylogenetic analysis of OsCESA7.

<p>(A) Multiple alignments of the N-terminal region of 11 members of the OsCESA family and 8 members of the AtCESA family. The mutated residue (cysteine 40) is highly conserved. (B) Phylogenetic analysis of CESAs. The scale bar is an indicator of genetic distance based on branch length.</p

Characterization of a Null Allelic Mutant of the Rice <i>NAL1</i> Gene Reveals Its Role in Regulating Cell Division

<div><p>Leaf morphology is closely associated with cell division. In rice, mutations in <i>Narrow leaf 1 (NAL1)</i> show narrow leaf phenotypes. Previous studies have shown that <i>NAL1</i> plays a role in regulating vein patterning and increasing grain yield in <i>indica</i> cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of <i>NARROW LEAF1 (NAL1)</i>, <i>nal1-2</i> and <i>nal1-3</i>, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that <i>nal1-2</i> is a null allelic mutant of <i>NAL1</i> since both the whole promoter and a 404-bp fragment in the first exon of <i>NAL1</i> were deleted, and that a 6-bp fragment was deleted in the mutant <i>nal1-3</i>. We demonstrated that <i>NAL1</i> functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that <i>NAL1</i> affects cell cycle regulation. Heterogenous expression of <i>NAL1</i> in fission yeast (<i>Schizosaccharomyces pombe</i>) further supported that <i>NAL1</i> affects cell division. These results suggest that <i>NAL1</i> controls leaf width and plant height through its effects on cell division.</p></div

Histological analyses of leaves.

<p>(A, B) The abaxial epidermis of the upper second leaf blade of wild-type (WT) (A) and <i>nal1-2</i> (B) plants (bars = 50 μm). (C–F) Cross sections through the middle part of the upper second leaf blade of WT (C, E) and <i>nal1-2</i> (D, F) plants. Red double-headed arrows indicate the thickness of the leaf mesophyll adjacent the midrib (C, D) and small veins (E, F). Red curves in C and D outline the parenchyma cell layers (bars = 50 μm). (G) Magnified images of the red-boxed regions in A (left) and B (right). Black arrows show the position of stomatal guard cells (bar = 50 μm). (H–L) Comparison of the leaf blade width (H), epidermal cell number (I), epidermal cell size (J), leaf mesophyll thickness (K), and the number of mesophyll cell layers (L) between the WT and <i>nal1-2</i>. Epidermal cell number was calculated by dividing each leaf blade width by the corresponding epidermal cell width. Data are shown as means ± standard error (SE) (H, I, K, and L, n ≥ 10; J, n ≥ 500). Student’s t-test was used to analyze significant differences between the WT and <i>nal1-2</i>. *, 0.01 ≤ P < 0.01; **, P < 0.01.</p