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1.
Trinucleotide repeats are common within gene coding regions and could serve as beacons to locate genes. Five of the most common
trinucleotide repeats in an Actinidia (kiwifruit) expressed sequence tag (EST) database were found to be (ACC)4, (CAC)4, (CCA)4, (CTC)4, and (TGG)4. These repeats, with or without an artificial 5′-end tail, were tested by vectorette PCR against genomic DNA from Actinidia chinensis. Eighty-nine randomly selected clones showed an average insert size of 383 bp, with a maximum of 1,151 bp and a minimum of
78 bp. Two-thirds of the clones contained the artificial tail attached to the trinucleotide, showing a slight advantage of
possessing such a tail during annealing and amplification. The sequences were searched against the Actinidia EST database and GenBank. Of the 89 clones, 33 had a significant hit (expect value < e−15). Twenty-four of those clones matched an Actinidia EST. Twenty-one clones contained one or more simple sequence repeats. This methodology can be applied by conventional cloning
and sequencing methods or by high throughput pyrosequencing technologies to develop genetic markers and also for gene mining
in species with little or no genetic/genomic resources. 相似文献
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Expressed sequence tags from a NaCl-treated Suaeda salsa cDNA library 总被引:15,自引:0,他引:15
Past efforts to improve plant tolerance to osmotic stress have had limited success owing to the genetic complexity of stress responses. The first step towards cataloging and categorizing genetically complex abotic stress responses is the rapid discovery of genes by the large-scale partial sequencing of randomly selected cDNA clones or expressed sequence tags (ESTs). Suaeda salsa, which can survive seawater-level salinity, is a favorite halophytic model for salt tolerant research. We constructed a NaCl-treated cDNA library of Suaeda salsa and sequenced 1048 randomly selected clones, out of which 1016 clones produced readable sequences (773 showed homology to previously identified genes, 227 matched unknown protein coding regions, 16 anomalous sequences or sequences of bacterial origin were excluded from further analysis). By sequence analysis we identified 492 unique clones: 315 showed homology to previously identified genes, 177 matched unknown protein coding regions (101 of which have been found before in other organisms and 76 are completely novel). All our EST data are available on the Internet. We believe that our dbEST and the associated DNA materials will be a useful source to scientists engaging in stress-tolerance study. 相似文献
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S. Ok Y. S. Chung B. Y. Um M. S. Park J.-M. Bae S. J. Lee J. S. Shin 《Plant cell reports》2000,19(9):932-937
A cDNA library was constructed using mRNA prepared from leaves of watermelon [Citrullus lanatus (Thunb.) Matsum&Nakai] at the vegetative stage. Randomly selected cDNA clones were sequenced in order to identify potentially
informative genes. Database comparisons indicated that out of the 704 watermelon cDNA clones, 399 clones (56.7 %) revealed
a high degree of sequence similarity to genes from other organisms. These expressed sequence tag clones were divided into
ten categories depending upon gene function. Since this kind of experiment has not previously been carried out in this genome,
random nucleotide sequencing of these cDNAs could contribute considerable information concerning the novel genes in this organism.
Received: 10 July 1999 / Revision received: 20 December 1999 / Accepted: 11 January 2000 相似文献
5.
Joonyul Kim Sung-Hoon jun Jinwon Lee Hong-Gyu Rang Gynheung An 《Journal of Plant Biology》2001,44(3):172-177
To understand the molecular events that occur during reproductive organ development and to provide genetic resources for molecular
breeding, we generated 328 expressed sequence tags (ESTs) from randomly selected clones of four watermelon cDNA libraries.
These libraries were prepared from young and mature anthers, as well as the seed coat and inner seed tissues. EST clones found
in the young anthers and inner seed tissues showed similarity with genes related to development and signal transduction. We
could deduce that, especially in the developing inner seed tissues, important morphological processes were associated exclusively
with seed and embryo development In addition, seed metabolism was tailored toward the accumulation of economically valuable
storage compounds such as lipids. In the seed coat, EST clones showed similarity with genes that influence the transport or
conversion of nutrients such as porin, sucrose synthase, L-asparaginase, and arginine decarboxylase. We also selected two
cDNA clones from each of the four classes of ESTs for studying expression levels and patterns in the various organs. Among
those eight clones, three (An88, Is124, and Sc68) were expressed preferentially in their particular organ. 相似文献
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The XIST gene plays an essential role in X Chromosome (Chr) inactivation during the early development of female humans. It
is believed that the XIST gene, not encoding a protein, functions as an RNA. The XIST cDNA is unusually long, as its full
length is reported to be 16.5 kilobase pairs (kb). Here, comparison of sequences from the genomic interval downstream to the
3′ end of the human XIST gene against the human EST database brought to light a number of human EST sequences that are mapped
to the region. Furthermore, PCR amplification of human cDNA libraries and RNA fluorescence in situ hybridization (RNA-FISH)
demonstrate that the human XIST gene has additional 2.8 kb downstream sequences which have not been documented as a part of
the gene. These data show that the full-length XIST cDNA is, in fact, 19.3 kb, not 16.5 kb as previously reported. The newly
defined region contains an intron that may be alternatively spliced and seven polyadenylation signal sequences. Sequences
in the newly defined region show overall sequence similarity with the 3′ terminal region of mouse Xist, and three subregions exhibit quite high sequence conservation. Interestingly, the new intron spans the first two subregions
that are absent in one of the two isoforms of mouse Xist. Taken together, we revise the structure of human XIST cDNA and compare cDNA structures between human and mouse XIST/Xist.
Received: 3 August 1999 / Accepted: 15 November 1999 相似文献
9.
Philippe Goyette Aditya Pai Renate Milos Phyllis Frosst Pamela Tran Zhoutao Chen Manuel Chan Rima Rozen 《Mammalian genome》1998,9(8):652-656
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate,
a co-substrate for homocysteine remethylation to methionine. A human cDNA for MTHFR, 2.2 kb in length, has been expressed
and shown to result in a catalytically active enzyme of approximately 70 kDa. Fifteen mutations have been identified in the
MTHFR gene: 14 rare mutations associated with severe enzymatic deficiency and 1 common variant associated with a milder deficiency.
The common polymorphism has been implicated in three multifactorial diseases: occlusive vascular disease, neural tube defects,
and colon cancer. The human gene has been mapped to chromosomal region 1p36.3 while the mouse gene has been localized to distal
Chromosome (Chr) 4. Here we report the isolation and characterization of the human and mouse genes for MTHFR. A human genomic
clone (17 kb) was found to contain the entire cDNA sequence of 2.2 kb; there were 11 exons ranging in size from 102 bp to
432 bp. Intron sizes ranged from 250 bp to 1.5 kb with one exception of 4.2 kb. The mouse genomic clones (19 kb) start 7 kb
5′ exon 1 and extend to the end of the coding sequence. The mouse amino acid sequence is approximately 90% identical to the
corresponding human sequence. The exon sizes, locations of intronic boundaries, and intron sizes are also quite similar between
the two species. The availability of human genomic clones has been useful in designing primers for exon amplification and
mutation detection. The mouse genomic clones will be helpful in designing constructs for gene targeting and generation of
mouse models for MTHFR deficiency.
Received: 28 January 1998 / Accepted: 9 April 1998 相似文献
10.
Hiroyuki Jikuya Jun Takano Reiko Kikuno Makoto Hirosawa Takahiro Nagase Nobuo Nomura Osamu Ohara 《DNA research》2003,10(1):49-57
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. 相似文献
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C S Keith D O Hoang B M Barrett B Feigelman M C Nelson H Thai C Baysdorfer 《Plant physiology》1993,101(1):329-332
As part of a project to identify novel maize (Zea mays L. cv B73) genes functionally, we have partially sequenced 130 randomly selected clones from a maize leaf cDNA library. Data base comparisons revealed seven previously sequenced maize cDNAs and 18 cDNAs with sequence similarity to related maize genes or to genes from other organisms. One hundred five cDNAs show little or no similarity to previously sequenced genes. Our results also establish the suitability of this library for large-scale sequencing in terms of its large insert size, proper insert orientation, and low duplication rate. 相似文献
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François Barrieu Dominique Thomas Danièle Marty-Mazars Maryse Charbonnier Francis Marty 《Planta》1998,204(3):335-344
The vacuolar membrane (tonoplast) of plant cells contains aquaporins, protein channels that facilitate the selective transport
of water. These tonoplast intrinsic proteins (TIPs) of 23–29 kDa belong to the ancient major intrinsic protein (MIP) family.
A monospecific polyclonal antiserum directed against a 26 kDa intrinsic protein from the tonoplast of meristematic cells from
cauliflower (Brassica oleracea L. var. botrytis) was used to screen a cDNA library. Two distinct cDNAs have been isolated. Both clones, c26-1 and c26-2, encode closely related TIPs. The c26-1 insert, consisting of 933 bp upstream of the poly(A) tail, is a full-length cDNA with an open reading frame encoding a protein
of 251 amino acids with a calculated Mr of 25 500. The c26-2 insert is a 5′ truncated cDNA. The two cDNAs share 90.5% sequence identity within their overlapping coding regions but only
35% sequence identity in the 3′␣untranslated regions, indicating that highly related TIP-encoding genes are expressed in meristematic
cells. Although TIPs have previously been found in a variety of cell types, they have not been found in meristems. The derived
amino acid sequences (BobTIP26-1 and BobTIP26-2, respectively) closely resemble the aquaporin γ-TIP from Arabidopsis thaliana. Northern blot analysis and in situ hybridization show that BobTIP26 mRNAs preferentially accumulate in highly meristematic cells, mostly before and during cell enlargement, and in the living
cells of the xylem. This differential pattern of expression is also found by immunodetection of BobTIP26 polypeptides. The
gene expression patterns are discussed with respect to the probable function of the gene products.
Received: 27 March 1997 / Accepted: 20 May 1997 相似文献
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Cheryl Frankfater Erika Maus Krisztina Gaal Fernando Segade Neal G. Copeland Debra J. Gilbert Nancy A. Jenkins J. Michael Shipley 《Mammalian genome》2000,11(3):191-195
A 1.4-kb EST clone encoding mouse microfibril-associated glycoprotein-2 (MAGP-2), identified by its similarity with the reported
human cDNA, was used to screen a mouse 129 genomic bacterial artificial chromosome (BAC) library. The mouse gene contains
10 exons spanning 16 kb, located on the distal region of Chromosome (Chr) 6. The exons range in size from 24 to 963 bp, with
the ATG located in exon 2. The tenth and largest exon contains 817 bp of 3′ untranslated sequence, including a B2 repetitive
element. Northern analysis demonstrates abundant expression of MAGP-2 mRNA in skeletal muscle, lung, and heart. Sequence analysis
of additional cDNA clones suggests that the two mRNA forms of MAGP-2 in the mouse arise from alternative polyadenylation site
usage. The promoter does not contain an obvious TATA box, and the sequence surrounding the start site does not conform to
the consensus for an initiator promoter element. Additionally, the mouse promoter contains 22 copies of a CT dinucleotide
repeat sequence located ∼155 bp 5′ to exon 1.
Received: 27 August 1999 / Accepted: 2 November 1999 相似文献
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G Uzan P Frachet A Lajmanovich M H Prandini E Denarier A Duperray J Loftus M Ginsberg E Plow G Marguerie 《European journal of biochemistry》1988,171(1-2):87-93
Platelet glycoprotein (GP) IIb is one of the two subunits of the common platelet adhesion receptor, GPIIb-IIIa. The isolation, characterization and sequencing of cDNA clones encoding for the two polypeptide chains of GPIIb are described. A number of clones were isolated from lambda gt11 libraries constructed with mRNA from an erythroleukemic cell line, HEL, and human megakaryocytes. Two of these clones, lambda IIb1, from HEL cells, and lambda IIb2, from megakaryocytes, cross-hybridized and were selected for detailed analysis. The identification of these as authentic GPIIb clones was based on immunological criteria and confirmed by the presence of nucleotide sequences in each insert encoding for known protein sequences of platelet GPIIb. These clones contained inserts of 1.54 kb and 1.39 kb, respectively, with an overlapping sequence of 801 bp. The nucleotide sequence of the overlapping region was identical indicating that HEL cells produce a protein closely related, if not identical, to platelet GPIIb. The determined nucleotide sequence of two inserts included a coding sequence for 648 amino acid residues, a TAG stop codon and 185 nucleotides of 3' non-coding sequence followed by a poly(A) tail. The coding sequence contained a portion of the heavy chain, the junction between the heavy and light chains and the entire light chain including a potential transmembrane-spanning domain and a short cytoplasmic tail. When these cDNA were used to probe for GPIIb mRNA, a single mRNA species of 3.9 kb was identified in both HEL cells and human megakaryocytes. A comparison of the deduced amino acid sequence for GPIIb with those of the alpha subunit of the vitronectin and the fibronectin receptors revealed extensive homologies. These homologies further establish that GPIIb-IIIa from platelets, together with the vitronectin and the fibronectin receptors, are members of a supergene family of adhesion receptors with a recognition specificity for Arg-Gly-Asp amino acid sequences. 相似文献
19.
We have isolated from a rat brain lambda gt11 expression library two overlapping cDNA clones of sizes 2.5 and 3.0 kb corresponding to the heavy neurofilament polypeptide (NF-H). The 2.5 kb insert apparently represents virtually the whole of the C-terminal tail, the 3.0 kb insert also encodes the conserved epitope for the monoclonal antibody, anti-IFA. The identity of the cDNAs was established by comparison of the predicted amino acid sequence with the known partial amino acid sequence of porcine NF-H. A repeat peptide sequence that may be a multiphosphorylation site was identified in the C-terminal non-helical tail. 相似文献
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Bianca Garcia Emilio Margolles Hernan Roca Dania Mateu Manuel Raices Maria Elena Gonzales Luis Herrera Julio Delgado 《FEMS microbiology letters》1996,143(2-3):175-183
Abstract A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-α-glucan hydrolase, EC 3.2.1.11) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8. 相似文献