The Genexpress Index: a resource for gene discovery and the genic map of the human genome.

Houlgatte, Rémi; Mariage‐Samson, Régine; Duprat, Simone; Tessier, Alan J.; Bentolila, S; Lamy, B.; Auffray, Charles

doi:10.1101/gr.5.3.272

Cited by 77 publications

(53 citation statements)

References 80 publications

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“…One of the ESTs for DYRK2 (GenBank TM accession number Z25301) has been mapped to human chromosome 12 in the Genexpress program (24). For DYRK3, the map position of an EST clone of the I.M.A.G.E.…”

Section: Identification Of Dyrk-related Kinases By Pcr Cloning-mentioning

confidence: 99%

Sequence Characteristics, Subcellular Localization, and Substrate Specificity of DYRK-related Kinases, a Novel Family of Dual Specificity Protein Kinases

Becker¹,

Weber²,

Wetzel³

et al. 1998

Journal of Biological Chemistry

267

276

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DYRK1 is a dual specificity protein kinase presumably involved in brain development. Here we show that the kinase belongs to a new family of protein kinases comprising at least seven mammalian isoforms (DYRK1A, DYRK1B, DYRK1C, DYRK2, DYRK3, DYRK4A, and DYRK4B), the yeast homolog Yak1p, and the Drosophila kinase minibrain (MNB). In rat tissues, DYRK1A is expressed ubiquitously, whereas transcripts for DYRK1B, DYRK2, DYRK3, and DYRK4 were detected predominantly in testes of adult but not prepuberal rats. By fluorescence microscopy and subcellular fractionation, a green fluorescent protein (GFP) fusion protein of DYRK1A was found to accumulate in the nucleus of transfected COS-7 and HEK293 cells, whereas GFP-DYRK2 was predominantly detected in the cytoplasm. DYRK1A exhibited a punctate pattern of GFP fluorescence inside the nucleus and was co-purified with the nuclear matrix. Analysis of GFP-DYRK1A deletion constructs showed that the nuclear localization of DYRK1A was mediated by its nuclear targeting signal (amino acids 105-139) but that its characteristic subnuclear distribution depended on additional N-terminal elements (amino acids 1-104). When expressed in Escherichia coli, DYRK1A, DYRK2, DYRK3, MNB, and Yak1p catalyzed their autophosphorylation on tyrosine residues. The kinases differed in their substrate specificity in that DYRK2 and DYRK3, but not DYRK1A and MNB, catalyzed phosphorylation of histone H2B. The heterogeneity of their subcellular localization and substrate specificity suggests that the kinases are involved in different cellular functions.

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Section: Identification Of Dyrk-related Kinases By Pcr Cloning-mentioning

confidence: 99%

Sequence Characteristics, Subcellular Localization, and Substrate Specificity of DYRK-related Kinases, a Novel Family of Dual Specificity Protein Kinases

Becker¹,

Weber²,

Wetzel³

et al. 1998

Journal of Biological Chemistry

267

276

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“…Complete sequencing of the inserts revealed three clone categories: 3-OST-2, I.M.A.G.E. Consortium (Lawrence Livermore National Laboratory) Clone ID c-20d10 (GenBank™ accession number F07258) (14) from a normalized library generated from total brain of a 3-month muscular atrophy female (15); 3-OST-3 A CTF , Clone ID 284542 (GenBank™ accession number N71828), from a library of 4 multiple sclerosis lesions isolated from a 46-year-old male (13); and 3-OST-4, Clone IDs HIBCX69 (GenBank™ accession number T33472) from human brain (16), IB727 (GenBank™ accession number T03677) from infant brain (17,18), 166466 (GenBank™ accession number R88592) from adult brain (13), 23279 (GenBank™ accession number T75445) from infant brain, c-3ie01 (GenBank™ accession number F13088) (from the same library as c-20d10) (14). To obtain full-length clones, we first identified cDNA regions, described below, which would function as isoform-specific probes by hybridizing Southern blots of human genomic DNA with expressed sequence tag fragments 32 P-labeled by random priming.…”

Section: Isolation Of 3-ost-2 3-ost-3 and 3-ost-4 Cdnamentioning

confidence: 99%

Multiple Isoforms of Heparan Sulfate d-Glucosaminyl 3-O-Sulfotransferase

Shworak

Liu

Petros

et al. 1999

Journal of Biological Chemistry

222

121

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3-O-Sulfated glucosaminyl residues are rare constituents of heparan sulfate and are essential for the activity of anticoagulant heparan sulfate. Cellular production of the critical active structure is controlled by the ratelimiting enzyme, heparan sulfate D-glucosaminyl 3-Osulfotransferase-1 (3-OST-1) (EC 2.8.2.23). We have probed the expressed sequence tag data base with the carboxyl-terminal sulfotransferase domain of 3-OST-1 to reveal three novel, incomplete human cDNAs. These were utilized in library screens to isolate full-length cDNAs. Clones corresponding to predominant transcripts were obtained for the 367-, 406-, and 390-amino acid enzymes 3-OST-2, 3-OST-3 A , and 3-OST-3 B , respectively. These type II integral membrane proteins are comprised of a divergent amino-terminal region and a very homologous carboxyl-terminal sulfotransferase domain of ϳ260 residues. Also recovered were partial length clones for 3-OST-4. Expression of the full-length enzymes confirms the 3-O-sulfation of specific glucosaminyl residues within heparan sulfate (Liu, J., Shworak, N. W., Sinaÿ , P., Schwartz, J. J. Zhang, L., Fritze, L. M. S., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, 5185-5192). Southern analyses suggest the human 3OST1, 3OST2, and 3OST4 genes, and the corresponding mouse isologs, are single copy. However, 3OST3A and 3OST3B genes are each duplicated in humans and show at least one copy each in mice. Intriguingly, the entire sulfotransferase domain sequence of the 3-OST-3 B cDNA (774 base pairs) was 99.2% identical to the same region of 3-OST-3 A . Together, these data argue that the structure of this functionally important region is actively maintained by gene conversion between 3OST3A and 3OST3B loci. Interspecific mouse back-cross analysis identified the loci for mouse 3Ost genes and syntenic assignments of corresponding human isologs were confirmed by the identification of mapped sequence-tagged site markers. Northern blot analyses indicate brain exclusive and brain predominant expression of 3-OST-4 and 3-OST-2 transcripts, respectively; whereas, 3-OST-3 A and 3-OST-3 B isoforms show widespread expression of multiple transcripts. The reiteration and conservation of the 3-OST sulfotransferase domain suggest that this structure is a self-contained functional unit. Moreover, the extensive number of 3OST genes with diverse expression patterns of multiple transcripts suggests that the novel 3-OST enzymes, like 3-OST-1, regulate important biologic properties of heparan sulfate proteoglycans.Heparan sulfate proteoglycans are hybrid molecules composed of a protein core to which is attached one or more linear glycosaminoglycan chains of the heparan sulfate variety. Extreme structural diversity of the heparan sulfate side chains enables interactions with a broad array of protein effector molecules that modulate a wide range of biologic processes. The specificity of any given heparan sulfate-protein interaction is largely dictated by placement of sulfate groups along the chain's length. Thus the order and ring posit...

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“…Accordingly, once most mRNAs of the prevalent and intermediate frequency classes are identified, redundancy levels are expected to become greater than 60%. For this reason, the use of normalized libraries, in which the frequency of all clones is within a narrow range , has been shown to be beneficial for largescale sequencing (Berry et al 1995;Houlgatte et al 1995). Calculations show that at Co t = 5.5 [where Co is the total DNA concentration and t is the time (moles nucleotides per liter • sec)], of the three kinetic classes of mRNAs, the most abundant species are diminished drastically, while all frequencies are brought within the range of one order of magnitude .…”

mentioning

confidence: 99%

Normalization and subtraction: two approaches to facilitate gene discovery.

Bonaldo¹,

Lennon²,

Soares³

1996

Genome Res.

491

356

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Large-scale sequencing of cDNAs randomly picked from libraries has proven to be a very powerful approach to discover (putatively) expressed sequences that, in turn, once mapped, may greatly expedite the process involved in the identification and cloning of human disease genes. However, the integrity of the data and the pace at which novel sequences can be identified depends to a great extent on the cDNA libraries that are used. Because altogether, in a typical cell, the mRNAs of the prevalent and intermediate frequency classes comprise as much as 50-65% of the total mRNA mass, but represent no more than 1000-2000 different mRNAs, redundant identification of mRNAs of these two frequency classes is destined to become overwhelming relatively early in any such random gene discovery programs, thus seriously compromising their cost-effectiveness. With the goal of facilitating such efforts, previously we developed a method to construct directionally cloned normalized cDNA libraries and applied it to generate infant brain (INIB) and fetal liver/spleen (INFLS) libraries, from which a total of 45,192 and 86,088 expressed sequence tags, respectively, have been derived. While improving the representation of the longest cDNAs in our libraries, we developed three additional methods to normalize cDNA libraries and generated over 35 libraries, most of which have been contributed to our integrated Molecular Analysis of Genomes and Their Expression (IMAGE) Consortium and thus distributed widely and used for sequencing and mapping. In an attempt to facilitate the process of gene discovery further, we have also developed a subtractive hybridization approach designed specifically to eliminate (or reduce significantly the representation of) large pools of arrayed and (mostly) sequenced clones from normalized libraries yet to be (or just partly) surveyed. Here we present a detailed description and a comparative analysis of four methods that we developed and used to generate normalize cDNA libraries from human (15), mouse (3), rat (2), as well as the parasite Schistosoma mansoni (1). In addition, we describe the construction and preliminary characterization of a subtracted liver/spleen library (INFLS-SI) that resulted from the elimination (or reduction of representation) of -5000 INFLS-IMAGE clones from the INFLS library.

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The Genexpress Index: a resource for gene discovery and the genic map of the human genome.

Cited by 77 publications

References 80 publications

Sequence Characteristics, Subcellular Localization, and Substrate Specificity of DYRK-related Kinases, a Novel Family of Dual Specificity Protein Kinases

Sequence Characteristics, Subcellular Localization, and Substrate Specificity of DYRK-related Kinases, a Novel Family of Dual Specificity Protein Kinases

Multiple Isoforms of Heparan Sulfate d-Glucosaminyl 3-O-Sulfotransferase

Normalization and subtraction: two approaches to facilitate gene discovery.

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