An Expression Profile of Active Genes in Human Lung

An expression profile ofgenes active in the human lung was obtainedby collecting 797 partial sequences from a 3'-directed cDNAlibrary. Three genes were found to produce mRNA each of whichcomprised more than 1% of total mRNA. These three have beenidentified as genes for pulmonary surfactant apoprotein (PSP-A),Clara cells 10-kDa secretory protein, and HLA-E heavy chain.In the remaining 745 clones, 221 were composed of89 speciesthat occurred recurrently, and 524 clones appeared only once.Because the 3'-directed cDNA library faithfully represents themRNA population in the source tissue, these numbers representthe relative activities ofthe gene expression. Altogether 437gene species were novel, and 179 gene species were identifiedin GenBank. A significant portion ofthese genes encode proteinsfound in secretory proteins, cell surface proteins, and componentsin the protein synthesis machinery, representing the functionof the lung.     

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.     

Gene Expression Profiling of Coccolith-Bearing Cells and Naked Cells in Haptophyte <Emphasis Type="Italic">Pleurochrysis haptonemofera</Emphasis> with a cDNA Macroarray System

Pleurochrysis haptonemofera is a unicellular marine coccolithophorid that has calcified scales, coccoliths, on the cell surface. Some coccolithophorids including P. haptonemofera have a coccolith-bearing stage and a naked stage in their life cycles. To characterize genes involved in the coccolithogenesis, we generated a total of 9550 expressed sequence tags (EST) from a normalized cDNA library that was prepared using both coccolith-bearing cells (C-cells) and naked cells (N-cells), constructed a cDNA macroarray using the EST clones, and then analyzed the gene expression specificity in C-cells and N-cells. When cDNA clones whose expression ratio exceeded 3-fold were selected, as many as 180 clones were identified as C-cell-specific ones, while only 12 were found to be N-cell-specific ones. These clones were sequenced, assembled, and homology-searched against a public nonredundant protein database. As a result, they were grouped into 54 C-cell-specific and 6 N-cell-specific genes, and 59% and 50% of these genes exhibited significant similarity to those of other known proteins, respectively. To assess mRNA expression further, Northern hybridization was performed for 12 of the C-cell-specific genes and one of the N-cell-specific ones. These clones, together with the new cDNA macroarray, will provide a powerful tool for the future genome-wide functional analysis of uncharacterized genes related to the regulation of the calcification and life cycle of coccolithophorids. Shoko Fujiwara and Yasutaka Hirokawa contributed equally to this work.     

Construction and Characterization of a Goat Mammary Gland cDNA Library

A lactating goat mammary gland cDNA library was constructed by using a modified commercially available cDNA library construction kit protocol. The resulting clones were sequenced and functionally analyzed through cross-species genomic comparison to assess (1) the capacity and functional quality of the constructed library for subsequent research and (2) the efficiency of the procedural modifications. The study resulted in the construction of a high-quality mammary gland cDNA library, which was characterized by (1) the total recombinants number of 1.4 × 10⁷ colony-forming units (cfus) that was at least 10 times greater than the number expected from the application of the standard kit protocol, (2) the recombinants rate of 96%, and (3) the average insert size of 1,082 bp. BLAST analysis of sequenced clones against GenBank databases determined 55.7% of clone redundancy, 22 known function gene clusters, and 29 novel gene clusters. The analysis of the primary gene expression profile showed that 59% of the tested clones were genes that coded for milk proteins while 16% of the clones coded for ribosomal, metabolism, immune response, and translation proteins. The remaining 25% of the tested clones were described as novel genes. Cross-species comparison showed that 77% of characterized gene clusters were successfully identified by using resources from other ruminants and unrelated species. This outcome is in consonance with the common belief that the genomic resources that have been generated across species are potentially powerful tools that could be used for enhancing the molecular understanding of less genomically studied species, such as goat.     

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.     

Genes active in developing wheat endosperm

This paper describes the construction and characterisation of a cDNA library from wheat endosperm tissue during the early stages of grain filling. Developing wheat endosperm tissue was characterised with respect to standard measures including dry weight, cytological appearance and timing of expression of major sources of mRNA such as the seed storage protein genes. In addition, the full complement of proteins present at mid-endosperm development was examined using 2D-electrophoretic techniques. Based on this characterisation, endosperm from the developing grain 8–12 days post-anthesis was chosen for isolating mRNA and preparing cDNA. At this stage in development the mRNA population is not yet dominated by the accumulation of mRNA from seed storage protein genes. A cDNA library, not normalised, containing a high percentage of full length cDNA clones was constructed and 4,319 clones sequenced ("single-pass"). Partitioning of the cDNA sequences into gene families and singletons provided the basis for quantifying the accumulation of sequence classes relative to the total number of sequences determined. The accumulation of gene families/singletons was not linear. However, mathematical modeling of the data suggested that the maximum number of different genes expressed is within the range of 4,500–8,000 (detailed in the Appendix). If an average is taken of these extremes, approximately 27% of the gene products were visible as proteins in the 2D-electrophoretic analysis. Analysis of a functional class of genes relevant to wheat grain end-use, namely the glutenin/gliadin seed storage protein class of genes, revealed a new category of gene characterised by a distinctive N-terminal domain and a reduced central repetitive domain. Electronic Publication     

Sequences of 596 cDNA Clones (565,977 bp) of Japanese Flounder (Paralichthys olivaceus) Leukocytes Infected with Hirame Rhabdovirus

We have partially sequenced 785 sequences of 596 independent complementary DNA clones isolated from a cDNA library of Japanese flounder leukocytes infected with hirame rhabdovirus. These sequences consist of a total of 565,977 base pairs. The average size of the sequenced lengths was 721 bp. Of 596 clones, 386 (64.8%) were identified as previously reported genes by the BLASTN and BLASTX programs. About 30% of the identified clones could be recognized by only the BLASTX program. A total of 251 distinct genes were identified, and 181 of these genes are the first such genes reported from the teleostei. Approximately 27% of the identified Japanese flounder genes appear to be associated with cell division, cell structure or motility, and basic energy metabolism, 29% with gene or protein expression, and 17% with cell signaling, cell communication, and cell or organism defense. The most frequently identified expressed sequence tags of leukocytes of Japanese flounder were gelatinase b and ribosomal protein L23, which both had 1.34% prevalence. Received February 12, 1999; accepted April 13, 1999     

Group 1 CD1 genes in rabbit

CD1 is an Ag-presenting molecule that can present lipids and glycolipids to T cells. The CD1 genes were first identified in the human, and since then, homologs have been identified in every mammalian species examined to date. Over a decade ago, CD1B and CD1D homologs were identified in the rabbit. We have extended this earlier study by identifying additional CD1 genes with the goal of developing the rabbit as an animal model to study the function of CD1 proteins. We constructed a thymocyte cDNA library and screened the library with CD1-specific probes. Based on nucleotide sequence analyses of the CD1(+) cDNA clones obtained from the library, we have identified two CD1A genes and one CD1E gene as well as determined the complete sequence of the previously identified CD1B gene. The CD1E(+) cDNA clones lacked the transmembrane and cytoplasmic domains and, if translated, would encode for a soluble or secreted CD1E protein. In addition, expression studies demonstrated that the CD1 genes were expressed in peripheral lymphoid tissues as well as in skin, gut, and lung. Of interest is the finding that CD1A2, CD1B, and CD1E genes were found to be expressed by rabbit B cell populations. The rabbit, with a complex CD1 locus composed of at least two CD1A genes, one CD1B gene, one CD1D gene, and one CD1E gene, is an excellent candidate as an animal model to study CD1 proteins.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

Expression of differentiated functions in the developing porcine small intestine

Heterologous cDNA clones were used as hybridization probes to define the temporal expression of intestinal functions during fetal and postnatal development in the pig. Northern hybridization analysis revealed the presence of the mRNAs for the cellular retinol binding protein CRBP II, for the digestive enzyme aminopeptidase N, and for the microvillar proteins villin and ezrin in the small intestine of both weaned and 40-day fetal pigs. The presence of these mRNAs suggests that at the end of the first third of gestation the pig fetal intestine is already exhibiting some characteristics of a differentiated epithelium. The mRNAs for the two fatty acid-binding proteins I-FABP and L-FAPB, both involved in the metabolism of long chain fatty acids, were detected only in the intestinal mRNA extracted from weaned animals, while that for the cellular retinol-binding protein CRBP I was expressed only in the fetal tissue. The temporal limits of expression of intestinal genes in the pig epithelium seem therefore more easily defined than in other experimental animals with shorter times of fetal development. To isolate pig genes expressed at different developmental stages during intestinal epithelial cell differentiation, a cDNA library was constructed from poly(A) + RNA extracted from mature pig intestine. This library was employed in the isolation of clones encoding CRBP II and L-FABP. The nucleotide sequence of the two pig cDNA clones was determined, and the sequences of the deduced proteins compared with their homologues from other species. The results of this analysis showed that the two pig clones share a high level of homology with human and rat homologues both at the DNA and at the protein level.     

Analysis of randomly isolated cDNAs from developing endosperm of rice (Oryza sativa L.): evaluation of expressed sequence tags,and expression levels of mRNAs

Using a cDNA library prepared from poly(A)⁺ RNA from 10-day-old rice endosperm, partial nucleotide sequences of randomly isolated clones were analyzed. A total of 153 (30.6%) out of 500 cDNA clones showed high amino acid identity to previously identified genes. There was significant redundancy in cDNAs encoding prolamine and glutelin. About 21.0% of the cDNA clones were found to code for seed storage protein genes. Consequently, 37 independent genes were identified. Using cDNA clones encoding glutelin, prolamine, seed allergen, -1,4-glucan branching enzyme, glycine-rich RNA binding protein, metallothionein, non-specific lipid-transfer protein and ubiquitin conjugating enzyme the accumulation of mRNA during rice seed development was compared. Genes associated with seed storage protein and starch biosynthesis were expressed according to expected developmental stages. Glycinerich RNA binding protein genes as well as metallothionein-like protein genes were highly expressed in developing seeds, but low in leaves of whole plants.     

高山植物黄管秦艽cDNA文库构建与表达序列标签（EST）分析

黄管秦艽（Gentiana officinalis）是一种重要的藏药高山植物,本研究构建了该物种开花期的eDNA文库。经检测达到中等cDNA文库水平,文库滴度为1．2×10^7pfu／ml,重组率95．9％,插入片段平均长度大于500bp。对343个随机挑选的重组克隆进行部分测序,获得的ESTs经编辑后共有181条有效序列。经生物信息学方法分析181条表达序列标签（EST）代表144个单克隆序列,其中55个与已鉴定的基因同源,35个序列与未鉴定的EST匹配,54个未找到同源序列;后两者共有89个EST序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现,相关基因主要编码以下蛋白：与蛋白表达相关的占35％;光合作用相关的占笠％;新陈代谢相关的占18％;抗性相关的占11％;质膜运输和细胞分裂相关的分别占5％;染色体变化和细胞信号转导的分别占2％。根据有效EST序列设计引物,通过RT-PCR进一验证了所得EST的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。     

Isolation of cold stress-responsive genes from Lepidium latifolium by suppressive subtraction hybridization

Suppression subtraction hybridization (SSH) libraries were constructed from RNA isolated from leaves of control and cold stress-induced Lepidium latifolium, a cold-tolerant plant species from high altitudes for isolation of cold-responsive genes. A total of 500 clones were obtained from the cold stress library. Dot blot expression analysis identified 157 clones that were upregulated and 75 that were downregulated during cold stress. These clones selected on the basis of their expression patterns on dot blot were sequenced. As much as 27 and 17 genes were identified from the forward and reverse libraries, respectively. The genes identified revealed homology with genes involved in diverse processes such as gene regulation/signaling, photosynthesis, DNA damage repair protein, pathogenesis-related protein, senescence-associated proteins and proteins with unknown functions.