Characterization of Overexpressed cDNAs Isolated from a Hormone-Autonomous, Radiation-Induced Tumor Tissue Line of Arabidopsis thaliana

To investigate the molecular mechanisms of hormonal control of growth, we constructed a subtracted cDNA library enriched for sequences expressed more in a hormone-autonomous, radiation-induced tumor tissue line of Arabidopsis thaliana than in normal, hormone-dependent callus. Ten cDNA clones, which are expressed 1.3- to 10-fold more in the tumor line, were isolated and partially characterized. The clones differ greatly in their level of expression in tumor tissue and in their pattern of expression in plant organs. Southern blot hybridization and sequence analysis showed that this group contains three pairs of closely related clones. Northern blot analysis indicates that one pair of clones represents two members of a gene family that are expressed in different plant organs. One of the isolated sequences shows strong sequence similarity to a cDNA encoding a lipid transfer protein. Two sequences are highly similar to those of previously described membrane channel proteins but have different organ specificities. Two other cDNAs have significant sequence similarity to glycine-rich proteins and hydroxy-proline-rich glycoproteins. When used to probe Southern blots, none of the cDNAs identified polymorphisms between tumor and callus DNA, which might be expected if their overexpression were due to local genome rearrangements induced by radiation. The diversity observed among these 10 clones suggests that some are likely to be involved in tumorous growth and not simply specific to a certain cell or tissue type present in the tumor.     

Restricted utilization of germ-line VH genes and diversity of D regions in rabbit splenic Ig mRNA

Previous studies have suggested that the majority of rabbit germ-line VH genes encode molecules that are rarely found in serum or secretory Ig. To examine the repertoire of expressed VH genes, we prepared a cDNA library from splenic mRNA of an alpha 1/alpha 1 rabbit and isolated 10 complete VH-encoding cDNA clones. None of the cDNA clones hybridized to an oligomer that had hybridized to more than 50% of cloned germ-line VH genes. These data indicate that only a subset of germ-line VH genes are used in functional VDJ rearrangements. DNA sequence analysis demonstrated that the 10 cDNA clones contained highly similar VH regions, further suggesting that the repertoire of utilized VH genes is limited. In contrast, the D regions of each of the 10 clones exhibited little similarity to one another, suggesting that the rabbit has a large D region repertoire. We propose that the apparent lack of diversity within the VH segment of VDJ rearrangements is offset by extensive D region diversity.     

Construction of two ordered cDNA libraries enriched in genes encoding plasmalemma and tonoplast proteins from a high-efficiency expression library

We constructed a high-efficiency expression library from Arabidopsis cDNA clones by introducing a poly (dC) stretch at the 5' end of the clones. This library enables the synthesis of proteins from all the cDNA clones present. We have screened the high-efficiency expression library with antibodies raised against total proteins from Arabidopsis plasmalemma and tonoplast. With the positive clones, we have constructed two cDNA ordered libraries enriched in genes encoding plasmalemma (522 clones) and tonoplast proteins (594 clones). Partial sequencing of both libraries shows that a high proportion (47%) of the clones encoded putative membrane proteins, or membrane-associated proteins. When sequenced, 55% of the cDNAs were new EST sequences for Arabidopsis, 26% were similar to genes present in other plants or organisms, and 29% were not referenced in any databank. Immunoscreening of the two cDNA ordered libraries with antibodies raised against proteins from Arabidopsis cells submitted to osmotic stress allows the selection of genes over- and under-expressed in stress conditions.     

The expression of an extensin-like protein correlates with cellular tip growth in tomato

Extensins are abundant proteins presumed to determine physical characteristics of the plant cell wall. We have cloned a cDNA encoding LeExt1 from a tomato (Lycopersicon esculentum Mill.) root hair cDNA library. The deduced sequence of the LeExt1 polypeptide defined a novel type of extensin-like proteins in tomato. Patterns of mRNA distribution indicated that expression of the LeExt1 gene was initiated in the root hair differentiation zone of the tomato rhizodermis. Cloning of the corresponding promoter and fusion to the -glucuronidase (GUS) reporter gene allowed detailed examination of LeExt1 expression in transgenic tomato plants. Evidence is presented for a direct correlation between LeExt1 expression and cellular tip growth. LeExt1/GUS expression was detectable in trichoblasts (=root hair-bearing cells), but not in atrichoblasts of the tomato rhizodermis. Both hair formation and LeExt1 expression was inducible by the plant hormone ethylene. Comparative analysis of the LeExt1/GUS expression was performed in transgenic tomato, potato (Solanum tuberosum), tobacco (Nicotiana tabacum), and Arabidopsis plants. In the apical/basal dimension, GUS staining was absent from the root cap and undifferentiated cells at the root tip in all species investigated. It was induced at the distal end of the differentiation zone and remained high proximally to the root/hypocotyl boundary. In the radial dimension, GUS expression was root hair specific in the solanaceous species. Whereas LeExt1 mRNA was exclusively detectable in the rhizodermis, root hair-specific expression correlated with GUS expression in germinating pollen tubes. This is correlative evidence for a role of LeExt1 in root hair tip growth [corrected].     

Expressed sequence tag analysis of expression profiles of zebrafish testis and ovary

In the present study, two gonad cDNA libraries from zebrafish testes and ovaries were constructed and a total of 1025 expressed sequence tag (EST) clones were generated from the two libraries: 501 from the testis library and 524 from the ovary library. A total of 641 of the EST clones were identified to share significant sequence identity with known sequences in GenBank, representing at least 478 different zebrafish genes. In order to understand the molecular compositions of the two gonad organs, the expression profiles of the identified clones in these two gonad cDNA libraries were analyzed. Both gonad libraries have a higher portion of clones for nuclear proteins and a lower portion for proteins in translational machinery, cytoskeleton and mitochondria than our previously characterized whole-adult cDNA library. Most abundant cDNA clones in the two gonad libraries were identified and over 10% of ovary clones were found to encode egg membrane proteins (zona pellucida or ZP proteins). Furthermore, the testis library showed a more even distribution of cDNA clones with relatively fewer abundant clones that tend to contribute redundant clones in EST projects; thus, the testis library can supply more unique and novel cDNA sequences in a zebrafish EST project. Another aim of this study is to identify cDNA clones that can be used as molecular markers for the analysis of the gonad development in zebrafish. Eleven potential clones were selected to analyze their expression patterns by Northern blot hybridization. Most of them showed a specific or predominant expression in the expected testis or ovary tissue. At last, four of the clones were found, by section in situ hybridization, to be expressed specifically in the germ cells of the testis or ovary and thus they are suitable molecular markers for analyses of spermatogenesis and oogenesis.     

Use of differential display for the identification of touch-induced genes from an ethylene-insensitive Arabidopsis mutant and partial characterization of these genes

Touch has been shown to affect plant growth and development and ethylene has been shown to have similar effects. However, the mechanisms responsible for touch-induced responses remain unclear. Differential display PCR was used to identify touch-regulated genes from 3-week-light-grown ethylene-insensitive etr1-3 Arabidopsis (Columbia ecotype) mutant plants. The differential display PCR screening process yielded 32 cDNA fragments. Subsequent screening of the 32 fragments using northern analysis yielded three touch-inducible clones (A8A, G5A and G7F). These three cDNA were then used to screen a cDNA library. A 1.2 kb fragment for OPR3 was obtained from A8A screenings. This cDNA fragment encodes 12-oxophytodienoate-10, 11-reductase (OPR), an enzyme in the jasmonic acid biosynthetic pathway. OPR3 was found to be induced by touch, wounding, methyl jasmonate (MeJA), NaCl and CaCl₂ while ethylene and darkness had no effect. A 2 kb cDNA encoding a calcium-dependent protein kinase (CDPK32) was obtained with G5A screenings. CDPK32 was shown to be induced by touch, wounding, NaCl and darkness while ethylene and MeJA had little or no effect. A 1.4 kb cDNA encoding a novel protein was recovered from the cDNA library screenings with a G7F fragment. This cDNA had some sequence similarity to GDA1 and was designated GDL for GDA1-like cDNA. GDL was activated by touch, wounding, MeJA, NaCl and CaCl₂ while there was no induction with ethylene and darkness. Using differential display PCR we have successfully been able to identify three clones that are inducible by touch and not by ethylene.     

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.     

Efficient identification of phosphatidylserine-binding proteins by ORF phage display

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in ∼300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.     

Multiple cDNA sequences and the evolution of bovine stomach lysozyme

To investigate the origin of stomach expression of lysozyme in ruminants; we surveyed clones from a cow stomach cDNA library with a lysozyme cDNA probe. Ten percent of the clones in this library were lysozyme-specific. Thirty of the lysozyme clones were sequenced, and seven types of lysozyme mRNA sequence were found. They encode the three previously identified stomach isozymes of lysozyme. The seven sequences are closely related to one another and represent the products of a minimum of 4 of the approximately 10 cow lysozyme genes detected by genomic blotting. The most abundant form of stomach lysozyme (form 2) is encoded by at least two genes, whereas forms 1 and 3 are possibly each encoded by only one gene. The number of genes encoding each isozyme appears to contribute the largest factor in the relative abundance of each isozyme. The multiple lysozyme genes expressed in the cow stomach are the result of gene duplications that occurred during ruminant evolution. The recruitment of lysozyme as a major enzyme in the stomach may thus have involved an early regulatory event and a later 4-7-fold increase in expression allowed by gene amplification. During this period, the amino acid sequences of these lysozymes have been evolving more slowly than those of nonruminant lysozymes.     

Purification and molecular cloning of chemosensory proteins from Bombyx mori

Soluble low molecular weight acidic proteins are suspected to transport stimulus molecules to the sensory neurons within insect sensilla. From the antennae of Bombyx mori, we have purified and sequenced a protein (BmorCSP1) bearing sequence similarity to a class of soluble chemosensory proteins recently discovered in several orders of insects. Based on its N-terminal sequence, the cDNA encoding this protein has been amplified and cloned. Differential screening of a B. mori antennal cDNA library led to the identification of a second gene encoding a related protein (BmorCSP2), sharing 35-40% identity to BmorCSP1 and chemosensory proteins from other species. The predicted secondary structures of moth's, chemosensory proteins comprise alpha-helical foldings at conserved positions and a reduced hydrophobicity with respect to this novel family of chemosensory proteins.     

金柑花蕾酵母双杂交cDNA文库构建及评价

为了探索金柑花发育的分子机制并研究与金柑花发育相关重要基因编码的蛋白质之间的相互作用,本研究以融安金柑不同生长时期的花蕾为材料,采用SMART技术构建了cDNA文库,对该文库进行了鉴定评价。经检测,所构建的cDNA文库滴度为2.83×10^8cfu/mL,文库库容为1.42×10^10cfu,重组率为97.91%,插入的双链cDNA片段长度主要分布在250~2 000 bp之间。结果表明,该文库达到了建库标准,理论上涵盖了全部与融安金柑花蕾发育相关的基因,为后续利用酵母双杂交技术筛选互作蛋白提供了物质基础。     

Genes encoding a group of related small secreted proteins from the gut of Hessian fly larvae [Mayetiola destructor （Say）]

A group of related genes has been isolated and characterized from the gut of Hessian fly larvae [Mayetiola destructor （Say）]. Members in this group appear to encode proteins with secretary signal peptides at the N-terminals. The mature putative proteins are small, acidic proteins with calculated molecular masses of 14.5 to 15.3 kDa, and isoelectric points from 4.56 to 4.88. Northern blot analysis revealed that these genes are expressed predominantly in the gut of Hessian fly larvae and pupae. Two related genes, GIOK1 and GIOK2, were isolated as tandem repeats. Both genes contain three exons and two introns. The intron/exon boundaries were conserved in terms of amino acid encoding, suggesting that they arose by gene duplication. The fact that the frequency of this group of clones in a gut cDNA library higher than that of total cDNA clones encoding digestive enzymes suggested that this group of proteins may perform an important function in the gut physiology of this insect. However, the exact functions of these proteins are as yet known since no sequence similarity could be identified between these proteins and any known sequences in public databases using standard methods.     

Cloning of a cDNA fragment encoding part of the protein moiety of the 58-kDa fibrinogen-binding mannoprotein of Candida albicans

Immunoscreening of a Candida albicans expression library with antibodies against the 58 kDa fibrinogen-binding mannoprotein (mp58) of the fungus resulted in the isolation of clones encoding the protein moiety of this molecule. Sequence of the 0.9 kb cDNA of one of the clones selected for further analysis, revealed an open reading frame coding for 292 amino acids, which displays sequence similarity to proteins belonging to a family of immunodominant antigens of Aspergillus spp. The gene corresponding to this cDNA was named FBP1 (fibrinogen-binding protein). These results represent the first report on the identification of C. albicans genes encoding surface receptors for host proteins.     

Identification of putative parasitism genes expressed in the esophageal gland cells of the soybean cyst nematode Heterodera glycines

Cloning parasitism genes encoding secretory proteins expressed in the esophageal gland cells is the key to understanding the molecular basis of nematode parasitism of plants. Suppression subtractive hybridization (SSH) with the microaspirated contents from Heterodera glycines esophageal gland cells and intestinal region was used to isolate genes expressed preferentially in the gland cells of parasitic stages. Twenty-three unique cDNA sequences from a SSH cDNA library were identified and hybridized to the genomic DNA of H. glycines in Southern blots. Full-length cDNAs of 21 clones were obtained by screening a gland-cell long-distance polymerase chain reaction cDNA library. Deduced proteins of 10 clones were preceded by a signal peptide for secretion, and PSORT II computer analysis predicted eight proteins as extracellular, one as nuclear, and one as plasmalemma localized. In situ hybridization showed that four of the predicted extracellular clones were expressed specifically in the dorsal gland cell, one in the subventral gland cells, and three in the intestine in H. glycines. The predicted nuclear clone and the plasmalemma-localized clone were expressed in the subventral gland cells and the dorsal gland cell, respectively. SSH is an efficient method for cloning putative parasitism genes encoding esophageal gland cell secretory proteins that may have a role in H. glycines parasitism of soybean.     

Growth suppression of Escherichia coli by induction of expression of mammalian genes with transmembrane or ATPase domains

Growth inhibition of Escherichia coli host cells is frequently observed when some mammalian genes are induced to express exogenously. To find common features of these mammalian genes, an assay was designed for the isolation of these genes which show growth-inhibitory effect on E. coli by induction of expression. Of 38,000 clones derived from a mouse brain cDNA library, 64 cDNA clones were systematically selected out by this method, of which 45 clones had putative open reading frames encoding proteins with putative membrane-associated regions or ATP-binding/ATPase activities. These results show that a fraction of membrane-associated proteins or ATP-binding/ATPase genes can be isolated from cDNA libraries by our simple method.