Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain

We isolated mouse cDNA encoding a novel FGF (251 amino acids). As this is the 23rd documented FGF, we termed it FGF-23. FGF-23 has a hydrophobic amino terminus ( approximately 24 amino acids), which is a typical signal sequence. As expected, recombinant mouse FGF-23 was efficiently secreted by High Five insect cell-infected recombinant baculovirus containing the cDNA, indicating that FGF-23 is a secreted protein. We also isolated human cDNA encoding FGF-23 (251 amino acids), which is highly identical ( approximately 72% amino acid identity) to mouse FGF-23. Of human FGF family members, FGF-23 is most similar to FGF-21 and FGF-19 ( approximately 24% and approximately 22% amino acid identities, respectively). Human FGF-23 gene was localized on the chromosome 12p13 and found to be tandem linked (within 5.5 kb) to human FGF-6 gene. The expression of FGF-23 mRNA in mouse adult tissues was examined by real-time quantitative polymerase chain reaction. FGF-23 mRNA was mainly expressed in the brain and thymus at low levels. The localization of FGF-23 mRNA in the brain was examined by in situ hybridization. FGF-23 mRNA in the brain was found to be preferentially expressed in the ventrolateral thalamic nucleus. Therefore, FGF-23 is expected a unique FGF that plays roles in the function of the ventrolateral thalamic nucleus.     

Genomic organization and tissue-specific expression of splice variants of mouse organic anion transporting polypeptide 2

cDNAs that code for mouse organic anion transporting polypeptide 2 (oatp2) have been cloned. At least three forms of mouse oatp2 cDNAs containing the same coding sequence were isolated. The common coding sequence is for a protein of 670 amino acids with 12 putative transmembrane domains. The deduced amino acid sequence of the mouse oatp2 shares 89% identity with the reported rat oatp2. Cloning and analysis of mouse oatp2 gene indicates that these isoforms are alternatively spliced products from the same gene. Heterogeneity was observed in the 5'-untranslated region of the cDNAs. Two of the three isoforms lacked the noncoding exon 3 sequence. Northern-blot hybridization analysis using the exon 3-specific probes demonstrated that mouse oatp2 mRNA containing exon 3 sequence is expressed in heart and lung, whereas exon 1-, 2-, and 17-specific probes detected mRNA only in brain and liver. The mouse oatp2 gene consists of 17 exons, including three noncoding exons, and 16 introns. All of the introns are flanked by GT-AG splice sequences except for intron 10 that is flanked by GC-AG splice sequence.     

Molecular cloning and characterization of mouse cardiac junctate isoforms.

Junctate is a newly identified integral ER/SR membrane calcium binding protein, which is an alternative splicing form of the same gene generating aspartyl beta-hydroxylase and junctin. Screening a mouse heart cDNA library using canine junctin cDNA as a probe yielded three complete mouse heart cDNAs. One of the cDNAs is homologous to the previously reported human junctate. The three mouse junctate proteins are composed of 270, 259, and 215 amino acids (we named them junctate-1, -2, and -3). The apparent molecular masses of the mouse junctates in SDS-PAGE were in the range between 40 and 53 kDa. Northern and Western blot analyses indicate that mouse junctates are expressed in heart, brain, spleen, lung, liver, kidney, and stomach, but not in skeletal muscle. The apparent molecular weights of junctates from heart and brain were somewhat different from those from the other tissues tested, suggesting that there are tissue-specific expression patterns of the different junctate isoforms. Immunohistochemical studies showed that junctates were expressed both in ventricular and atrial tissues. This is the first study that shows the presence of 3 distinct cardiac junctate isoforms expressed in various mammalian tissues.     

Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing.

Using anchored PCR, three different cDNA isoforms of the mouse retinoic acid receptor beta [mRAR-beta 1, mRAR-beta 2 (formerly mRAR-beta 0) and mRAR-beta 3], generated from the same gene by differential promoter usage and alternative splicing, were isolated. These three isoforms encode RAR proteins with different N-terminal A regions and identical B - F regions. The sequence encoding the first 59 amino acids of the mRAR-beta 3 A region is identical with the entire A region of mRAR-beta 1. However, the sequence of mRAR-beta 3 region A differs from that of mRAR-beta 1 by an additional 27 C-terminal amino acids encoded in an 81 nucleotide-long putative exon which is spliced in between the exons encoding the A and B regions of mRAR-beta 1. Both mRAR-beta 1 and beta 3 cDNAs differ entirely from mRAR-beta 2 in their 5'-untranslated (5'-UTR) and A region coding sequences. This N-terminal variability, in a region which was shown to be important for cell-type specific differential target gene trans-activation by other nuclear receptors, suggests that the three mRAR-beta isoforms may be functionally distinct. The conservation of RAR-beta isoform sequences from mouse to human, as seen by cross-hybridization on Southern blots or DNA sequence analysis, as well as their differential patterns of expression in various mouse tissues, corroborates this view. Additionally, the mRNA analysis data suggest that mRAR-beta 2, whose expression predominates in RA-treated embryonal carcinoma (EC) and embryonic stem (ES) cells, may be important during early stages of development. mRAR-beta 1 and beta 3, on the other hand, which are predominantly expressed in fetal and adult brain, may play some specific role in the development of the central nervous system.     

Four fibroblast tropomyosin isoforms are expressed from the rat alpha-tropomyosin gene via alternative RNA splicing and the use of two promoters

cDNA clones encoding four rat tropomyosin isoforms, termed TM-2, TM-3, TM-5a, and TM-5b, were isolated and characterized. All are derived from the alpha-tropomyosin gene via alternative RNA processing and the use of two alternate promoters. The cDNA sequences predict that TM-2 and TM-3 both contain 284 amino acids and differ from each other only at an internal region of the protein from amino acids 189 through 213, due to alternative splicing of exons 6a and 6b. TM-5a and TM-5b both contain 248 amino acids and differ from each other only at an internal exon encoding amino acids 153 through 177, also due to alternative splicing of exons 6a and 6b. The differences in the amino acid sequence encoded by these alternate exons affects the theoretical actin-binding pattern of the tropomyosins, such that TM-5b is expected to bind actin with greater affinity than TM-5a. TM-2 and TM-3 are transcribed from the upstream promoter, and TM-5a and TM-5b are transcribed from an internal promoter. In addition, all four isoforms contain the identical COOH-terminal coding region. RNA protection analyses revealed that the mRNA for each isoform is expressed in a number of different tissues and cell types, although the expression of some isoforms is restricted to particular cell types. Furthermore, the expression of mRNA encoding these isoforms was found to be altered in a number of different virally transformed cell lines. The changes in the expression of tropomyosin mRNAs in transformed cells reflect changes in the relative use of the two promoters, as well as the relative use of alternatively spliced exons 6a and 6b.     

优雅蝈螽水溶性和类膜结合型海藻糖酶编码cDNA的鉴定及其在体内的表达

海藻糖酶是一种二糖水解酶,催化海藻糖转换为葡萄糖,为昆虫包括发育、壳多糖合成及飞翔代谢在内的多种生理过程所必需。尽管某些昆虫的海藻糖酶基因已被鉴定,但优雅蝈螽的海藻糖酶编码序列尚未见报告。本研究采用RACE结合多重PCR技术,分离鉴定优雅蝈螽的水溶性海藻糖酶（GgTre1）和类膜结合型海藻糖酶（GgTre2-like）的全长编码序列（cDNA）,包括携带不同长度3′-非翻译区（3′-UTR）的3个GgTre1 cDNA 亚型（GenBank：No.KY400001-KY400003）和3个GgTre2-like cDNA 亚型（GenBank：No.KY400004-KY400006）。 3个GgTre1 cDNA序列分别为2 107,2 021和1 914 bp,具有相同长度的5′-UTR（33 bp）,但3′-UTR 长度不同,分别为322,248和129 bp。GgTre1-2 cDNA含1 740 bp,编码579 个氨基酸残基组成的多肽链,分子量为67.29 kD;与之不同,根据cDNA演绎的GgTre1-1和GgTre1-3序列较GgTre1-2多4个氨基酸残基,多肽链的分子量为67.88 kD。3个GgTre2-like cDNA（GgTre2-like-1,-2和-3）序列全长分别为2 491,2 460 和2 381 bp。5′-UTR 均为284 bp,3′-UTR 分别为398,367和285 bp。GgTre2-like cDNA开阅读框为1 809 bp,编码602 氨基酸残基组成的多肽链,分子量为67.88 kD。实时定量PCR, 分析GgTre1和GgTre2-like基因在雌、雄个体（各20个）的组织特异性表达。结果显示,GgTre1 在卵巢和附腺表达量最高;GgTre2-like 主要在卵巢表达,在雄性肌肉和马氏管的表达量高于其他组织。上述结果表明,本研究从优雅蝈螽分离到3′-UTR长度不同的3个水溶性和3个类膜结合型海藻糖酶cDNA序列。结果还提示,GgTre1 在各组织的表达差异较大,而GgTre2-like 在各组织的表达相对稳定。不同长度3′-UTR的GgTre1 和 GgTre2-like 亚型的存在,以及不同长度的3′-UTR在翻译过程中的特殊作用,尚待今后研究证实。     

A repressor element in the 5'-untranslated region of human Pax5 exon 1A

Five members of the RecQ helicase family, RECQL, WRN, BLM, RTS and RECQL5, have been found in human and three of them (WRN, BLM and RTS) were disclosed to be the genes responsible for Werner, Bloom and Rothmund–Thomson syndromes, respectively. RECQL5 (RecQ helicase protein-like 5) was isolated as the fifth member of the family in humans through a search of homologous expressed sequence tags. The gene is expressed with at least three alternative splicing products, , β and γ. Here, we isolated mouse RECQL5β and determined the DNA sequence of full-length cDNA as well as the genome organization and chromosome locus. The mouse RECQL5β gene consists of 2949 bp coding 982 amino acid residues. Comparison of amino acid sequence among human (Homo sapiens), mouse (Mus musculus), Drosophila melanogaster and Caenorhabditis elegans RECQL5β homologs revealed three portions of highly conserved regions in addition to the helicase domain. Nineteen exons are dispersed over 40 kbp in the genome and all of the acceptor and donor sites for the splicing of each exon conform to the GT/AG rule. The gene is localized to the mouse chromosome 11E2, which has a syntenic relation to human 17q25.2-q25.3 where human RECQL5β exists. Our genetic characterizations of the mouse RECQL5β gene will contribute to functional studies on the RECQL5β products.     

The neuronal glycine transporter GLYT2 associates with membrane rafts: functional modulation by lipid environment

The neuronal glycine transporter GLYT2 is a plasma membrane protein that removes the neurotransmitter glycine from the synaptic cleft, thereby aiding the pre-synaptic terminal reloading and the termination of the glycinergic signal. Missense mutations in the gene encoding GLYT2 (SLC6A5) cause hyperekplexia in humans. The activity of GLYT2 seems to be highly regulated. In this report, we demonstrate that GLYT2 is associated with membrane rafts in the plasma membrane of brainstem terminals and neurons. The transporter is localized to Triton X-100-insoluble light synaptosomal membranes together with flotillin-1, a marker protein for membrane rafts, in a methyl-β-cyclodextrin (MβCD)-sensitive manner. In brainstem primary neurons, the GLYT2 punctuate pattern visualized by confocal microscopy was modified by cholesterol depletion with MβCD, unlike other non-raft neuronal markers. GLYT2-associated gold particles were observed by electron microscopy on purified rafts from brainstem synaptosomes. Furthermore, either in brainstem terminals and cultured neurons, the pharmacological reduction of the levels of raft components, cholesterol and sphingomyelin, impairs both the association of GLYT2 with membrane rafts and its transport activity. Thus, GLYT2 may require membrane raft location for optimal function, and therefore the lipid environment may constitute a new mechanism to modulate GLYT2.     

Zn2+ inhibits glycine transport by glycine transporter subtype 1b

In the central nervous system, glycine is a co-agonist with glutamate at the N-methyl-D-aspartate subtype of glutamate receptors and also an agonist at inhibitory, strychnine-sensitive glycine receptors. The GLYT1 subtypes of glycine transporters (GLYTs) are responsible for regulation of glycine at excitatory synapses, whereas a combination of GLYT1 and GLYT2 subtypes of glycine transporters are used at inhibitory glycinergic synapses. Zn2+ is stored in synaptic vesicles with glutamate in a number of regions of the brain and is believed to play a role in modulation of excitatory neurotransmission. In this study we have investigated the actions of Zn2+ on the glycine transporters, GLYT1b and GLYT2a, expressed in Xenopus laevis oocytes and we demonstrate that Zn2+ is a noncompetitive inhibitor of GLYT1 but has no effect on GLYT2. We have also investigated the molecular basis for these differences and the relationship between the Zn2+ and proton binding sites on GLYT1. Using site-directed mutagenesis, we identified 2 histidine residues, His-243 in the large second extracellular loop (ECL2) and His-410 in the fourth extracellular loop (ECL4), as two coordinates in the Zn2+ binding site of GLYT1b. In addition, our study suggests that the molecular determinants of proton regulation of GLYT1b are localized to the 2 histidine residues (His-410 and His-421) of ECL4. The ability of Zn2+ and protons to regulate the rate of glycine transport by interacting with residues situated in ECL4 of GLYT1b suggests that this region may influence the substrate translocation mechanism.     

The human muscle nicotinic acetylcholine receptor alpha-subunit exist as two isoforms: a novel exon.

Analysis of acetylcholine receptor clones isolated from a human leg muscle cDNA library, revealed that the alpha-subunit existed as two isoforms. A novel exon, coding for 25 amino acids, was located in the human genomic DNA sequence; its insertion into the alpha-subunit gives the new isoform of 462 amino acids. In addition, mRNAs for the two isoforms were found in equal proportions in poly(A)+ RNA obtained from three further sources including partially denervated and innervated human muscle and the rhabdomyosarcoma cell line TE671. Both protein isoforms can be expressed in E. coli. No evidence of a sequence related to that of the new exon was found in cDNA derived from poly(A)+ RNA isolated from fetal calf or embryonic chick muscle or Torpedo marmorata electric organ.