Gene expression of the ericoid mycorrhizal fungus Oidiodendron maius in the presence of high zinc concentrations

A heavy metal tolerant strain of the ericoid mycorrhizal species Oidiodendron maius, isolated from roots of Vaccinium myrtillus growing in soil heavily contaminated with zinc, was previously shown to tolerate high concentrations of zinc and cadmium ions in the growth medium. We have investigated the genetic basis of this fungal strain tolerance to high zinc concentrations by using an untargeted approach. From a cDNA library constructed by using mRNA from Zn-treated O. maius mycelia, 444 clones were randomly selected and 318 were sequenced. Sequence analysis identified 219 unique clones: 117 showed homology to previously identified genes, 26 matched unknown protein coding regions found in other organisms, and 76 were novel. Variation in the gene expression level after a 20-day treatment with high concentrations of Zn was monitored on 130 unigenes by reverse northern blot hybridisation. Sixteen unigenes were shown to be either up- (9) or down- (7) regulated. The putative function of these genes and their involvement in stress tolerance is discussed.     

Assignment of 118 novel cDNAs of cynomolgus monkey brain to human chromosomes

In order to isolate genes that may not be represented in current human brain cDNA libraries, we have sequenced about 20,000 sequence tags of cDNA clones derived from cerebellum and parietal lobe of cynomolgus monkeys (Macaca fascicularis). We determined the entire cDNA sequence of approximately 700 clones whose 5'-terminal sequences showed no homology to annotated putative genes or expressed sequence tags in current databases of genetic information. From this, 118 clones with sequences encoding novel open reading frames of more than 100 amino acid residues were selected for further analysis. To localize the genes corresponding to these 118 newly identified cDNA clones on human chromosomes, we performed a homology search using the human genome sequence and fluorescent in situ hybridization. In total, 108 of 118 clones were successfully assigned to specific regions of human chromosomes. This result demonstrates that genes expressed in cynomolgus monkey are highly conserved throughout primate evolution, and that virtually all had human homologs. Furthermore, we will be able to discover novel human genes in the human genome using monkey homologs as probes.     

Partial sequence analysis of randomly selected watermelon [Citrullus lanantus (Thunb.) Mansf.] cDNA clones

An expressed sequence tag (EST) is simply a segment of a sequence over 150 bp from a randomly selected cDNA. EST helps to quickly identify functions of expressed genes and to understand the complexity of gene expression with database comparison. Sequencing of random cDNA clones can be applicable to discovery of new genes, mapping of the genome, identification of coding regions in genomic sequences, and antisense method. To accomplish these goals, in this research, randomly selected cDNA sequencing was performed with watermelon plant. Among 30 clones picked up and analyzed, all clones had an insert length over 0.5 kb. The average size of insert was about 1.3 kb. The size range of most cDNA insert was 1.0–2.0 kb. For sequence comparison, data from the coding region at 5′ end of selected cDNA should be much more informative than those from the untranslated 3′ tail. Thirty clones were sequenced from one end (5′ end). Of these, 29 had no poly (A) tail in this direction, while only one was inverted. Thus, this library is over 96% unidirectional. Two clones had homologies to ribulose bisphosphate carboxylase/oxigenase (Rubisco) small subunit precursor gene. Thirteen cDNAs had high degree of sequence similarity to genes from other organisms. The remaining cDNA clones seem to be new genes not only in watermelon but also in all organisms.     

Isolation of developmentally regulated novel genes based on sequence identity and gene expression pattern.

Based on the surmise that a variety of genes might play important roles in embryonic development and tissue differentiation, and that some of them are likely to be expressed in undifferentiated ES cells, we attempted to identify new genes from the ES cell cDNA library. The modified method of expressed sequence tags (ESTs) and the examination of the expression patterns in adult tissues and in vitro differentiated ES cells were utilized in this study. We have isolated and identified several novel cDNA clones with interesting developmental expression pattern. Among the 83 clones randomly chosen, 23 clones (27.7%) have no homology to any sequences in public databases. The rest contain limited or complete sequence homology to the previously reported mammalian genes or ESTs, yet some clones have not been previously identified in the mouse. To examine the expression profile of clones during development and differentiation, sets of slot blots were hybridized with developmental stage specific or tissue specific probes. Out of 40 novel clones tested (21 totally unknown clones and 19 unidentified clones in mouse), most of them were up- or down-regulated as differentiation proceeded, and some clones showed differentiation-stage specific expression profiles. Surprisingly, a majority of genes were also expressed in adult tissues, and some clones even revealed tissue specific expression. These results demonstrate that not only was the strategy we employed in this study quite efficient for screening novel genes, but that the information gained by such studies would also be a useful guide for further analysis of these genes. It also suggests the feasibility of this approach to explore the genomewide network of gene expression during complicated biological processes, such as embryonic development and tissue differentiation.     

Structural analysis of the three vitellogenin genes in Drosophila melanogaster.

Genomic fragments coding for sequences expressed as abundant mRNA in female Drosophila melanogaster were isolated from a lambda library. Hybridization of these clones to polytene chromosomes. in situ, identified four which mapped to X chromosomal region 9A to 9B, the locus for yolk proteins 1 and 2 (Ypl,2) and two which mapped to 12A6-7 to 12D3, the locus for Yp3. These clones were mapped with restriction enzymes, and the coding regions and regions of homology determined by Southern blots probed with cDNA, 5'-end-labelled RNA and nick-translated DNA. Heteroduplex and R-loop mapping confirmed that three of the clones carried two genes separated by about 1.4 kb and oriented in opposite directions. Southern blots probed with cDNA made from alkali-hydrolyzed RNA showed that these genes had their 5' ends next to each other. All 3 genes show homology to each other and have a main coding region of about 1.3 kb, the approximate size for the mRNAs.     

Analysis of Expressed Sequence Tags from Skeletal Muscle-specific cDNA Library of Chinese Native Xiang Pig

A Longissimus Dorsi muscle cDNA library of Xiang Pig was constructed, and 131 randomly isolated clones were sequenced in this study. The results of bioinformatics analysis showed that 131 ESTs represented 109 unique clones sequences, of which 99 showed homology to previously identified genes in humans or other mammals, 3 matched other uncharacterized expressed sequence tags (ESTs), and 7 showed no significant matches to sequences already present in DNA databases. No protein matches were found for 10 ESTs. Functional analysis of the ESTs showed that a considerable proportion of them encoded proteins involved in gene/protein expression (45.46%). Other classes included genes involved in metabolism (10.10%), cell structure/motility (10.10%), cell/organism defense (5.05%), cell signaling/communication (2.02%), and cell division (0.0%). Unclassified genes constituted the remaining 27.27%. This study reported the results of the first gene expression profile analysis of Chinese native Xiang Pig skeletal muscle cells, thereby greatly facilitating the functional study of candidate genes involved in muscle growth as well as in the improvement of meat quality in domestic pigs.     

Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility.     

Cloning and Differential Expression of Two Catalases in Suaeda salsa in Response to Salt Stress

Two different cDNA clones (Sscat1 and Sscat2) encoding catalase, the primary important H2O2_scavenging enzyme, were isolated from a λZap_cDNA library constructed from a 400 mmol/L NaCl_treated library of Suaeda salsa (L.) Pall aerial tissue. Sscat1 (1.7 kb) contains a full open reading frame of 492 amino acids and Sscat2 (1.1 kb) is a partial clone. BLAST analysis indicates that the two clones share 71.9% identity in nucleotide sequence and 75% identity in deduced amino acid sequence within the last 287 amino acid residues of Sscat1. Southern blotting analysis showed that Sscat1 is multicopy in S. salsa genome, while Sscat2 is a single copy gene. Northern blotting analysis showed a rapid increase in the steady_level of both genes in roots after 48 h salt treatment, but only Sscat1 was induced in salinity treated leaves. Time_course analysis carried out in leaves confirmed that Sscat1 was induced by salt stress, in contrast to Sscat2. These implied that the expression of Sscat1 and Sscat2 genes are differentially regulated in S. salsa. The activity of total catalase is dramatically increased in response to salt stress.     

Construction of Rice cDNA Library and Partial Sequence Analysis of 100 Randomly Selected cDNA

A cDNA library of rice (Oryza sativa ssp. indica cv. "Guangluai 4") etiolated shoot was constructed using Lambda ZAP Ⅱ vector. After analysing the partial sequences of 100 randomly selected clones and database comparison to rice and other plants, 13 % cDNA clones could be identified and 12 % cDNAs had high degree of sequence similarity to partial sequence from rice or other species, whose function is still unknown. The remaining 75% cDNAs showed little or no similarity to genes in the database and might represent novel genes. It demonstrates the suitability of this library for large-scale sequencing from which more information of functional genes will result.     

中国地方品种香猪的肌肉特异组织表达序列标签(ESTs)的

通过构建香猪肌肉组织cDNA文库,并在文库中随机挑选克隆进行测序的方法,获得了131个香猪肌肉EST序列.在这131个EST序列所代表的109个单一克隆中,有99个为人类及其他物种的同源序列,3个为已知的猪的ESTs,7个为未知ESTs.对这10个已知、未知ESTs进行开放阅读框预测并进行B1ast分析,没有找到高度同源的氨基酸序列.对上述EST所对应的基因功能分析结果表明,除去27.27%的EST未能分类外,克隆到的EST大多来自与基因/蛋白的表达调控相关的基因(占45.46%).来自具有其他功能的基因的EST依次是细胞代谢占10.10%、细胞结构/迁移占10.10%、细胞/机体防御占5.05%和细胞信号/传导占2.02%.没有发现和细胞分裂相关的已知功能基因.本研究结果为中国地方品种香猪提供了第一个骨骼肌的基因表达谱,为今后寻找猪肌肉生长和肉用品质的候选基因奠定了基础.