Ching-Tack Han

Repression of hspA2 messenger RNA in human testes with abnormal spermatogenesis

OBJECTIVE To evaluate the messenger RNA (mRNA) expression of hspA2 in testes of infertile men with azoospermia. DESIGN Prospective study. SETTING Center for Reproduction and Genetics, Pundang Je-Saeng General Hospital, Dae-Jin Medical Center, Korea. PATIENT(S) Azoospermic patients (n = 15) undergoing testicular biopsy for pathologic evaluation were selected. INTERVENTION(S) After pathologic evaluation, testicular biopsy specimens were subdivided into three groups: group 1, normal spermatogenesis (n = 5); group 2, spermatocyte arrest (n = 5); and group 3, Sertoli cell-only syndrome (n = 5). The levels of hspA2 mRNA expression were compared in testes of group 1, group 2, and group 3 with the use of a competitive reverse transcription polymerase chain reaction (RT-PCR) technique. MAIN OUTCOME MEASURE(S) Comparison of hspA2 mRNA levels in testes. RESULT(S) On competitive RT-PCR analyses for hspA2 mRNA, significant hspA2 expression was observed in group 1, whereas a very low level of hspA2 was expressed in groups 2 and 3. CONCLUSION(S) This study demonstrates that hspA2 gene expression is down-regulated in human testes with abnormal spermatogenesis, which in turn suggests that the hspA2 gene might play a specific role during meiosis in human testes.

Molecular characterization of the Brassica rapa auxin-repressed, superfamily genes, BrARP1 and BrDRM1.

Two auxin-repressed superfamily genes, auxin-repressed protein 1 (ARP1) and dormancy-associated protein 1 (DRM1), are highly expressed in both the dormant buds and non-growing tissues of several plant species. To further identify the function of these proteins in Chinese cabbage (Brassica rapa L. ssp. pekinensis), we examined comprehensive expression patterns of BrARP1 and BrDRM1 under various developmental and stress conditions. We also examined these same genes in transgenic Arabidopsis plants. Both genes were expressed in all tissues tested, but their levels were highest in mature tissues accompanied by low levels of the growth-associated marker, B. rapa ribosomal protein 27. Expression of both genes was induced by abiotic stresses, such as chilling, heat shock, and salt treatment. Overexpression of either BrARP1 or BrDRM1 in Arabidopsis causes a reduction in vegetative growth and seed productivity, without affecting morphology. The lengths of petioles and siliques were greatly reduced. Simultaneous expression of both genes showed an additive effect on the growth suppression, resulting in significant reduction in plant size. Knock-out of Arabidopsis ARP1, DRM1, or both, neither affected growth rate nor final size. Results suggest BrARP1 and BrDRM1 are either involved in growth arrest, or stop growth, possibly from inhibition of either cell elongation or cell expansion, thereby creating a “growth brake”.

Developmental expression patterns of alpha1H T-type Ca2+ channels during spermatogenesis and organogenesis in mice.

The objectives of the present study were to investigate the expression patterns of T‐type Ca2+ channel mRNA during spermatogenesis and organogenesis in mice. Reverse transcription–polymerase chain reaction (RT–PCR) was performed to identify the subtypes of calcium channels present in the round spermatids isolated from mouse testes by flow cytometry. Transcripts of L‐type (α1D), non‐L‐type (α1E) and T‐type Ca2+ channels were detected in round spermatids. Analysis of PCR products of T‐type Ca2+ channels indicated that only α1H subunits were detected in round spermatids. The appearance and differential distribution of α1H T‐type Ca2+ channel mRNA during mouse spermatogenesis and postimplantation embryogenesis (embryonic (E) days E9, E12, E15) were investigated by in situ hybridization with digoxigenin‐labeled RNA probes coupled with alkaline phosphatase detection. In testes from adult and immature mice (postnatal 2 and 3 weeks), α1H T‐type Ca2+ channel mRNA was expressed in all developing germ cells and sertoli cells. On E9 and E12, tissues of the central nervous system, such as the telencephalon, expressed α1H T‐type Ca2+ channel mRNA. On E15, signals were detected throughout all organs of the embryo. These findings indicate that the expression of α1H T‐type Ca2+ channels is spatio‐temporally regulated during spermatogenesis and organogenesis.

Genome-wide expression profiles of contrasting inbred lines of Chinese cabbage, Chiifu and Kenshin, under temperature stress

Abiotic stresses such as extreme temperatures frequently limit plant growth and productivity of major crop species. To identify temperature-stress responsive genes in Brassica rapa ssp. pekinensis, we conducted a comparative transcriptome analysis of two contrasting genotypes, the inbred lines Chiifu and Kenshin, under and heat-shock conditions. Of 23,929 unigenes deposited on a Br24K microarray, 17,603 genes were significantly expressed. Chiifu expression profiles were more prevalent under , and those of Kenshin were more prevalent under heat-shock. Previously characterized genes, with little exception, show similar changes to temperature stress. We found that a relatively large number of genes were expressed in response to temperature stress and genotype, amounting to 24 and 126 genes from and heat-shock conditions, respectively. These genes could be used in molecular marker development for either high or low temperature-resistant Brassica spp. Only ten genes, including TCH4 and CBL1, were up-regulated in both genotypes under all stress treatments, implying possible candidates for broad spectrum, stress-resistant crop production.

Isolation and characterization of cDNA clones expressed under male sex expression conditions in a monoecious cucumber plant (Cucumis sativus L. cv. Winter Long)

In order to clone sex determination genes, both differential screening of a cDNA library and differential display reverse transcription PCR (DDRT-PCR) were conducted, using apex tissues from a Korean cucumber cultivar that exhibited different sex expression responses to differing photoperiods. Two genes (CsM1 and CsM2) expressed only under specific male expression conditions were then cloned via differential screening of a cDNA library and designated CsCYR (Cucumis sativus cyclin-related) and CsCYP (C. sativus cyclophilin). Both genes appear to be single-copy genes, and both express under male-expressing SD (short-day) conditions. CsM10 (C. sativus male specific clone 10) was isolated via DDRT-PCR, and was expressed preferentially under male expression conditions. The CsM10 gene encodes a non-coding RNA, and harbors a 179bp-conserved sequence, found in three genes which have been classified as a biotic stress signal-associated non-translatable RNA (or non-coding RNA) group. The CsM10 gene is also a single-copy gene, which is localized in the telomeric region of chromosome 6, and its expression is different in different tissues, developmental stages, and photoperiods. Based on the obtained sequence structures and expression patterns of CsM10 and its homologues, we suggest that CsM10 function appears to occur at the RNA level.

GDSL esterase/lipase genes in Brassica rapa L.: genome-wide identification and expression analysis.

GDSL esterase/lipase proteins (GELPs), a very large subfamily of lipolytic enzymes, have been identified in microbes and many plants, but only a few have been characterized with respect to their roles in growth, development, and stress responses. In Brassica crops, as in many other species, genome-wide systematic analysis and functional studies of these genes are still lacking. As a first step to study their function in B. rapa ssp. pekinensis (Chinese cabbage), we comprehensively identified all GELP genes in the genome. We found a total of 121 Brassica rapa GDSL esterase/lipase protein genes (BrGELPs), forming three clades in the phylogenetic analysis (two major and one minor), with an asymmetrical chromosomal distribution. Most BrGELPs possess four strictly conserved residues (Ser-Gly-Asn-His) in four separate conserved regions, along with short conserved and clade-specific blocks, suggesting functional diversification of these proteins. Detailed expression profiling revealed that BrGELPs were expressed in various tissues, including floral organs, implying that BrGELPs play diverse roles in various tissues and during development. Ten percent of BrGELPs were specifically expressed in fertile buds, rather than male-sterile buds, implying their involvement in pollen development. Analyses of EXL6 (extracellular lipase 6) expression and its co-expressed genes in both B. rapa and Arabidopsis, as well as knockdown of this gene in Arabidopsis, revealed that this gene plays an important role in pollen development in both species. The data described in this study will facilitate future investigations of other BrGELP functions.

Global Gene-Expression Analysis to Identify Differentially Expressed Genes Critical for the Heat Stress Response in Brassica rapa.

Genome-wide dissection of the heat stress response (HSR) is necessary to overcome problems in crop production caused by global warming. To identify HSR genes, we profiled gene expression in two Chinese cabbage inbred lines with different thermotolerances, Chiifu and Kenshin. Many genes exhibited >2-fold changes in expression upon exposure to 0.5– 4 h at 45°C (high temperature, HT): 5.2% (2,142 genes) in Chiifu and 3.7% (1,535 genes) in Kenshin. The most enriched GO (Gene Ontology) items included ‘response to heat’, ‘response to reactive oxygen species (ROS)’, ‘response to temperature stimulus’, ‘response to abiotic stimulus’, and ‘MAPKKK cascade’. In both lines, the genes most highly induced by HT encoded small heat shock proteins (Hsps) and heat shock factor (Hsf)-like proteins such as HsfB2A (Bra029292), whereas high-molecular weight Hsps were constitutively expressed. Other upstream HSR components were also up-regulated: ROS-scavenging genes like glutathione peroxidase 2 (BrGPX2, Bra022853), protein kinases, and phosphatases. Among heat stress (HS) marker genes in Arabidopsis, only exportin 1A (XPO1A) (Bra008580, Bra006382) can be applied to B. rapa for basal thermotolerance (BT) and short-term acquired thermotolerance (SAT) gene. CYP707A3 (Bra025083, Bra021965), which is involved in the dehydration response in Arabidopsis, was associated with membrane leakage in both lines following HS. Although many transcription factors (TF) genes, including DREB2A (Bra005852), were involved in HS tolerance in both lines, Bra024224 (MYB41) and Bra021735 (a bZIP/AIR1 [Anthocyanin-Impaired-Response-1]) were specific to Kenshin. Several candidate TFs involved in thermotolerance were confirmed as HSR genes by real-time PCR, and these assignments were further supported by promoter analysis. Although some of our findings are similar to those obtained using other plant species, clear differences in Brassica rapa reveal a distinct HSR in this species. Our data could also provide a springboard for developing molecular markers of HS and for engineering HS tolerant B. rapa.

Overexpression of BrMORN, a novel ‘membrane occupation and recognition nexus’ motif protein gene from Chinese cabbage, promotes vegetative growth and seed production in Arabidopsis

Proteins that contain membrane occupation and recognition nexus (MORN) motifs regulate various aspects of cellular metabolism by localizing proteins in different cellular organelles. The full-length Brassica rapa MORN motif protein (BrMORN) cDNA consists of 1,510 bp encoding 502 deduced amino acids with a predicted molecular mass of 55.8 kDa and an isoelectric point of 9.72. BrMORN is a novel protein composed of two N-terminal transmembrane helices and seven C-terminal MORN motifs and it appears to be localized on the plastid envelope. BrMORN expression was relatively high in actively-growing tissues, but low in mature tissues and under some abiotic stresses. Arabidopsis thaliana plants overexpressing BrMORN showed an enhanced rate of growth, hypocotyl elongation, and increases in the size of vegetative organs and seed productivity under normal growth conditions. In addition, cell size in Arabidopsis plants overexpressing BrMORN was 24% larger than that of wild-type plants, implying that the increase in the size of vegetative organs is due to cell enlargement. The increased size of the vegetative organs also led to increased seed production. Our data suggest that the MORN motif of BrMORN may act at the plastid envelope and facilitate plant growth via cell enlargement.

Expression characteristics of heat shock protein genes in two comparable inbred lines of Chinese cabbage, Chiifu and Kenshin

Heat-shock proteins (HSPs) and heat-shock transcription factors (HSFs) are central components of the heat-shock regulatory network and are involved in cellular responses to various forms of stresses. To examine the differences in heat shock responses (HSRs) of two comparable inbred lines of Chinese cabbage (Brassica rapa), 51 genes were selected from 130,000 Brassica rapa ESTs that belong to an HSF and six HSP families and examined their expression using RT-PCR. Two Chinese cabbage inbred lines, Chiifu and Kenshin, have different geographic origins, in that Chiifu is from temperate regions, while Kenshin is from subtropical and tropical regions. Among the 51 genes, six genes were induced, eleven were stimulated, and three were reduced in both inbred lines in response to heat shock (HS) treatment. However, eleven genes were differentially expressed between the two inbred lines. Among these genes, several appear to be involved in normal growth and chloroplast development. These data suggest that the two Chinese cabbage inbred lines have similar HSRs, but the unique HSRs allow Kenshin to develop at higher temperatures.

Genome-wide analysis of genes associated with bolting in heading type chinese cabbage

Bolting or flowering time affects productivity of the leafy vegetable Chinese cabbage (Brassica rapa ssp. pekinensis) and also the time required for its breeding programs. Understanding the bolting process at the molecular level and identifying the genes involved will provide valuable tools for genetic engineering and development of functional markers. To achieve these goals, microarray analyses using either outer leaves or core tissues of completely headed Chinese cabbage were performed at three developmental stages, and the findings were confirmed using RT-PCR. A large number of genes were specifically or preferentially expressed in each tissue: photosynthesis and abiotic signal responding genes were expressed in outer leaves, while genes involved in hormone responses, flower development, and histone modification were expressed in core tissues. Genes promoting bolting, such as BrPIF4, BrPIF5, and BrCOLs, were highly expressed in outer leaves, a signal-perceiving tissue, whereas floral repressors, such as BrFLCs, BrFRL, and BrMAF1s, were predominantly expressed in core tissues, with levels of expression decreasing in later stages. Although the B. rapa genome contains three BrFT paralogs, only BrFT1 was expressed. BrFD and BrFE, genes related to the transport of BrFTs, showed consistent expression. The late bolting phenotype of Huissen, the F1 variety examined in this study, may result from expression of repressors of flowering, such as BrGASA5, BrTFL1, BrMAF1, BrFLCs, and BrKNAT1, in core tissues. Coordinated regulation of several floral promoters and repressors appears to be necessary for control of the bolting process or flowering time in B. rapa.