47个早期人胚胎低丰度表达基因ESTs筛选及结果分析

构建高质量ｃＤＮＡ文库在基因克隆、ｍＲＮＡ差异展示、表达序列标签测序和基因定位等研究中具有十分重要的作用。为了从早期胚胎中分离人类新基因,构建了受精后３周龄的人ｃＤＮＡ文库,用标记的一链ｃＤＮＡ探针对该文库的６５０８个克隆子进行菌落原位杂交,得到１６７７个无任何杂交信号的低丰度表达克隆子,从中随机挑选了４７个进行５′端部分测序,将测序结果与三大基因库进行序列同源性比较,发现１８个克隆（３８．３％）     

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.     

Expressed sequence tags of Chinese cabbage flower bud cDNA.

We randomly selected and partially sequenced cDNA clones from a library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower bud cDNAs. Out of 1216 expressed sequence tags (ESTs), 904 cDNA clones were unique or nonredundant. Five hundred eighty-eight clones (48.4%) had sequence homology to functionally defined genes at the peptide level. Only 5 clones encoded known flower-specific proteins. Among the cDNAs with no similarity to known protein sequences (628), 184 clones had significant similarity to nucleotide sequences registered in the databases. Among these 184 clones, 142 exhibited similarities at the nucleotide level only with plant ESTs. Also, sequence similarities were evident between these 142 ESTs and their matching ESTs when compared using the deduced amino acid sequences. Therefore, it is possible that the anonymous ESTs encode plant-specific ubiquitous proteins. Our extensive EST analysis of genes expressed in floral organs not only contributes to the understanding of the dynamics of genome expression patterns in floral organs but also adds data to the repertoire of all genomic genes.     

Construction and characterization of normalized cDNA library of maize inbred Mo17 from multiple tissues and developmental stages

Comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10.830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarry was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate gene, and developing microarrays in maize genomics research.     

Construction of expression-ready cDNA clones for KIAA genes: manual curation of 330 KIAA cDNA clones.

We have accumulated information on protein-coding sequences of uncharacterized human genes, which are known as KIAA genes, through cDNA sequencing. For comprehensive functional analysis of the KIAA genes, it is necessary to prepare a set of cDNA clones which direct the synthesis of functional KIAA gene products. However, since the KIAA cDNAs were derived from long mRNAs (> 4 kb), it was not expected that all of them were full-length. Thus, as the first step toward preparing these clones, we evaluated the integrity of protein-coding sequences of KIAA cDNA clones through comparison with homologous protein entries in the public database. As a result, 1141 KIAA cDNAs had at least one homologous entry in the database, and 619 of them (54%) were found to be truncated at the 5' and/or 3' ends. In this study, 290 KIAA cDNA clones were tailored to be full-length or have considerably longer sequences than the original clones by isolating additional cDNA clones and/or connected parts of additional cDNAs or PCR products of the missing portion to the original cDNA clone. Consequently, 265, 8, and 17 predicted CDSs of KIAA cDNA clones were increased in the amino-, carboxy-, and both terminal sequences, respectively. In addition, 40 cDNA clones were modified to remove spurious interruption of protein-coding sequences. The total length of the resultant extensions at amino- and carboxy-terminals of KIAA gene products reached 97,000 and 7,216 amino acid residues, respectively, and various protein domains were found in these extended portions.     

Construction and characterization of normalized cDNA library of maize inbred MO17 from multiple tissues and developmental stages

A comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10 830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarray was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using the BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate genes, and developing microarrays in maize genomics research.__________From Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 198–206.Original English Text Copyright © 2005 by Z. Zhang, F. Zhang, Tang, Pi, Zheng.This article was submitted by the authors in English.     

Identification of Alternaria brassicicola genes expressed in planta during pathogenesis of Arabidopsis thaliana

Alternaria brassicicola is a necrotrophic fungal pathogen that causes black spot disease on cruciferous plants including economically important Brassica species. The purpose of this study was to identify fungal genes expressed during infection of Arabidopsis. In order to identify candidate genes involved in pathogenicity, we employed suppression subtractive hybridization (SSH) between RNA isolated from A. brassicicola spores incubated in water and on the leaf surface of the Arabidopsis ecotype Landsberg. Two populations of cDNA were created from total RNA extracted after 24h when approximately 80% of the spores had germinated either on the leaf surface or in water. Following SSH, expression of clones was examined using dot-blot macro-arrays and virtual Northern blots. 47 cDNA clones differentially expressed between Alternaria infected Arabidopsis leaves and spore germination in water were selected for sequencing. Seventy-seven percent (36) of the cDNAs had significant homology to fungal sequences from databases examined, including available fungal genomes, while 13% (11) had no homology to sequences in the databases. All 36 genes had significant matches with genes of fungal origin, while 11 genes did not have significant hits in the databases examined. Five sequences were expressed on the plant leaf surface but not during spore germination in water according to virtual Northern blots. These five cDNAs were predicted to encode a cyanide hydratase, arsenic ATPase, formate dehydrogenase, major Alternaria allergen, and one unknown. RT-PCR was used to examine the expression of these five genes during infection of Brassica oleraceae var. capitata (cabbage), in vitro growth in nutrient rich media, and infection of Arabidopsis thaliana. Four of these genes are expressed in the nutrient rich medium, while the unknown gene P3F2 was only expressed during plant infection. The results of this study provide the first insight into genes expressed during A. brassicicola infection of Brassica species that may be involved in fungal pathogenesis.     

Discovery of 342 putative new genes from the analysis of 5'-end-sequenced full-length-enriched cDNA human transcripts

In this work we describe the process that, starting with the production of human full-length-enriched cDNA libraries using the CAP-Trapper method, led us to the discovery of 342 putative new human genes. Twenty-three thousand full-length-enriched clones, obtained from various cell lines and tissues in different developmental stages, were 5'-end sequenced, allowing the identification of a pool of 5300 unique cDNAs. By comparing these sequences to various human and vertebrate nucleotide databases we found that about 40% of our clones extended previously annotated 5' ends, 662 clones were likely to represent splice variants of known genes, and finally 342 clones remained unknown, with no or poor functional annotation. cDNA-microarray gene expression analysis showed that 260 of 342 unknown clones are expressed in at least one cell line and/or tissue. Further analysis of their sequences and the corresponding genomic locations allowed us to conclude that most of them represent potential novel genes, with only a small fraction having protein-coding potential.     

Feline arylsulfatase B (ARSB): isolation and expression of the cDNA, comparison with human ARSB, and gene localization to feline chromosome A1.

Arylsulfatase B (ARSB) is the lysosomal enzyme that catalyzes the hydrolysis of 4-sulfate groups from N-acetylgalactosamine 4-sulfate moieties on the glycosaminoglycans, dermatan sulfate and chondroitin sulfate A. In man, a deficiency of this enzymatic activity causes the lysosomal storage disorder, Maroteaux-Lamy disease (mucopolysaccharidosis Type VI; MPS VI). MPS VI in Siamese cats also has been described, and the comparative pathologic and biochemical abnormalities of the human and feline disorders have been well characterized. The present study describes the isolation and expression of cDNAs encoding feline ARSB and the assignment of the feline ARSB gene to feline chromosome A1. The full-length feline ARSB cDNA sequence is 1939 bp, including 3 and 328 bp of 5' and 3' untranslated sequences, respectively, and a 1608-bp open reading frame encoding 535 amino acids. The predicted human and feline ARSB proteins are 91% identical and 94% similar. However, despite this high homology, the predicted feline ARSB polypeptide has nine cysteine residues, while the human enzyme has eight. The presence of the extra cysteine residue at position 451 in the feline enzyme may explain why feline ARSB is a homodimer and the human enzyme is a monomer. To facilitate comparative structure/function studies of the human and feline enzymes and to initiate somatic gene therapy trials in the MPS VI cats, a full-length feline ARSB cDNA was reconstructed from a 1440-bp partial cDNA and an ARSB fragment amplified from feline first-strand cDNA by the polymerase chain reaction. The functional integrity of this cDNA was demonstrated by transient expression in human embryonic kidney cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and characterization of the family of feline leucine-rich glioma-inactivated (LGI) genes,and mutational analysis in familial spontaneous epileptic cats

Background

Leucine-rich glioma-inactivated (LGI) proteins play a critical role in synaptic transmission. Dysfunction of these genes and encoded proteins is associated with neurological disorders such as genetic epilepsy or autoimmune limbic encephalitis in animals and human. Familial spontaneous epileptic cats (FSECs) are the only feline strain and animal model of familial temporal lobe epilepsy. The seizure semiology of FSECs comprises recurrent limbic seizures with or without evolution into generalized epileptic seizures, while cats with antibodies against voltage-gated potassium channel complexed/LGI1 show limbic encephalitis and recurrent limbic seizures. However, it remains unclear whether the genetics underlying FSECs are associated with LGI family genes. In the present study, we cloned and characterized the feline LGI1–4 genes and examined their association with FSECs. Conventional PCR techniques were performed for cloning and mutational analysis. Characterization was predicted using bioinformatics software.

Results

The cDNAs of feline LGI1–4 contained 1674-bp, 1650-bp, 1647-bp, and 1617-bp open reading frames, respectively, and encoded proteins comprising 557, 549, 548, and 538 amino acid residues, respectively. The feline LGI1–4 putative protein sequences showed high homology with Homo sapiens, Canis familiaris, Bos taurus, Sus scrofa, and Equus caballus (92%–100%). Mutational analysis in 8 FSECs and 8 controls for LGI family genes revealed 3 non-synonymous and 14 synonymous single nucleotide polymorphisms in the coding region. Only one non-synonymous single nucleotide polymorphism in LGI4 was found in 3 out of 8 FSECs. Using three separate computational tools, this mutation was not predicted to be disease causing. No co-segregation of the disease was found with any variant.

Conclusions

We cloned the cDNAs of the four feline LGI genes, analyzed the amino acid sequences, and revealed that epilepsy in FSEC is not a monogenic disorder associated with LGI genes.

     

Primary structures and catalytic properties of isoenzymes encoded by the two 4-coumarate: CoA ligase genes in parsley

We have determined the primary structures of two 4-coumarate: CoA ligase (4CL) isoenzymes in parsley (Petroselinum crispum) by sequencing near full-length cDNAs corresponding to the two 4CL genes, Pc4CL-1 and Pc4CL-2, present in this plant. Comparison of the cDNA and genomic nucleotide sequences showed that each 4CL gene is organized in five exons separated by introns of varying lengths. The positions of introns are the same in both genes and 97-99% of the corresponding nucleotide sequences are identical. The two isoenzymes, which are nearly identical in their primary structures, were separated by ion-exchange chromatography, and were found to be indistinguishable with regard to substrate specificity. Assignment to Pc4CL-1 and Pc4CL-2 was achieved by comparison with catalytically active 4CL proteins, isolated from Escherichia coli cells which had been transformed with plasmids harboring the corresponding cDNAs.     

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.