Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family--oncogenic activation of v-kit involves deletion of extracellular domain and C terminus.

The protein kinase domains of v-kit, the oncogene of the acute transforming feline retrovirus HZ4-FeSV (HZ4-feline sarcoma virus), CSF-1R (macrophage colony stimulating factor receptor) and PDGFR (platelet derived growth factor receptor) display extensive homology. Because of the close structural relationship of v-kit, CSF-1R and PDGFR we predicted that c-kit would encode a protein kinase transmembrane receptor (Besmer et al., 1986a; Yarden et al., 1986). We have now determined the primary structure of murine c-kit from a DNA clone isolated from a brain cDNA library. The nucleotide sequence of the c-kit cDNA predicts a 975 amino acid protein product with a calculated mol. wt of 109.001 kd. It contains an N-terminal signal peptide, a transmembrane domain (residues 519-543) and in the C-terminal half the v-kit homologous sequences (residues 558-925). c-kit therefore contains the features which are characteristic of a transmembrane receptor kinase. Comparison of c-kit, CSF-1R and PDGFR revealed a unique structural relationship of these receptor kinases suggesting a common evolutionary origin. The outer cellular domain of c-kit was shown to be related to the immunoglobulin superfamily. The sites of expression of c-kit in normal tissue predict a function in the brain and in hematopoietic cells. N-terminal sequences which include the extracellular domain and the transmembrane domain as well as 50 amino acids from the C-terminus of c-kit are deleted in v-kit. These structural alterations are likely determinants of the oncogenic activation of v-kit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phylogenetic specificity of prolactin gene expression with conservation of Pit-1 function.

In mammals, the pituitary POU homeodomain protein, Pit-1, binds to proximal and distal 5'-flanking sequences of the PRL gene that dictate tissue-specific expression. These DNA sequences are highly conserved among mammals but are dramatically different from PRL 5' sequences in the teleost species, Oncorhynchus tschawytscha (chinook salmon). To analyze the molecular basis for pituitary-specific gene expression in a distantly related vertebrate, we transfected CAT reporter gene constructs containing 2.4 kilobases (kb) 5'-flanking sequence from the salmon PRL (sPRL) gene into various cell types. Expression of the sPRL gene was restricted to pituitary cells, but in rat pituitary GH4 cells levels of expression were at least 90-fold lower than those obtained with a -3 kb rat PRL (rPRL) construct. Conversely, in primary teleost pituitary cells, -2.4 kb sPRL/CAT was expressed at levels about 10-fold higher than -3 kb rPRL/CAT. To determine whether species-specific transactivation by Pit-1 was sufficient to explain these species differences in PRL gene expression, we isolated a cDNA clone encoding the salmon Pit-1 POU domain and constructed a rat Pit-1 expression vector that contained salmon Pit-1 POU domain sequences substituted in frame. The chimeric Pit-1 encoded 14 amino acids unique to salmon. Coexpression of rat Pit-1 with salmon or rat PRL/CAT in transfected HeLa cells resulted in specific and strikingly comparable levels of promoter activation. Moreover, the specificity and efficacy of the chimeric salmon/rat Pit-1 was similar to wild type rat Pit-1 in activating salmon and rat PRL/CAT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and characterization of N-syndecan, a novel transmembrane heparan sulfate proteoglycan

A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain.     

孔石莼(Ulva pertusa)质体蓝素基因的克隆及基因特征分析(英文)

采用cDNA末端快速扩增的办法,从孔石莼(Ulva pertusa)中克隆获得质体蓝素基因。该基因完整的cDNA为787bp,包括40 bp 5’端非编码区和327 bp的3’端非编码区,以及一个420 bp的开放阅读框架,编码139个氨基酸的蛋白质。该基因编码质体蓝素的前体肽,其N端41个氨基酸残基为信号肽,后面为98个氨基酸残基的成熟肽。从Genbank中选择了13个质体蓝素的前体肽基因进行序列比对分析和构建进化树。孔石莼质体蓝素基因与其它质体蓝素基因的同源性为48.2%至78.8%。该进化树将来源于6种藻类植物的7个质体蓝素基因聚类在一起,显示出它们较近的进化关系。同样,也表现出11种生物的分子进化关系。序列比对结果显示,在质体蓝素的基因序列中存在两个高度保守的基序,它编码质体蓝素蛋白的铜结合活性位点。     

Mu opioid receptor-like sequences are present throughout vertebrate evolution

The sequence of the mu opioid receptor is highly conserved among human, rat, and mouse. In order to gain insights into the evolution of the mu opioid receptor, polymerase chain reaction (PCR) was used to screen genomic DNA from a number of different species using degenerate oligonucleotides which recognize a highly conserved region. DNA was assayed from representative species of both the protostome and deuterostome branches of the metazoan phylogenetic tree. Mu opioid receptor-like sequences were found in all vertebrate species that were analyzed. These species included bovine, chicken, bullfrog, striped bass, thresher shark, and Pacific hagfish. However, no mu opioid receptor-like sequences were detected from protostomes or from any invertebrates. The PCR results demonstrate that the region of the mu opioid receptor gene between the first intracellular loop and the third transmembrane domain (TM3) has been highly conserved during evolution and that mu opioid receptor-like sequences are present in the earliest stages of vertebrate evolution. Additional opioid receptor-like sequence was obtained from mRNA isolated from Pacific hagfish brain using rapid amplification of cDNA ends (RACE). The sequence of the Pacific hagfish was most homologous with the human mu opioid receptor (72% at the amino acid level between intracellular loop 1 and transmembrane domain 6) although over the same region high homology was also observed with the delta opioid receptor (69%), the kappa receptor (63%), and opioid receptor-like (ORL1) (59%). The hagfish sequence showed low conservation with the mammalian opioid receptors in the first and second extracellular loops but high conservation in the transmembrane and intracellular domains. Received: 5 January 1996 / Accepted: 7 March 1996     

心脏形成关键基因-TBX5在大鼠胚胎心脏中的克隆及表达研究

为了研究心脏形成关键基因-TBX5的表达情况,选择Wistar大鼠作为从分子水平研究心脏发育的实验动物并从胎鼠心脏中克隆了TBX5cDNA全长（GenBank登录号：AY859491）,其cDNA及氨基酸序列不同于GenBank预测序列;随后应用RT-PCR及Northem blot分析TBX5基因在大鼠各组织中的表达情况,发现TBX5为单一转录本,在大鼠各组织均有表达,心脏中表达最强;构建荧光表达载体,转染大鼠肝癌细胞,观察TBX5亚细胞定位,结果证实TBX5基因在细胞核中表达;最后构建原核表达载体,IPTG诱导表达获得了GST-TBX5融合蛋白。以上工作为进一步鉴定心脏发育中TBX5相关转录因子及研究相互间的调控机制奠定了基础。     

花胫绿纹蝗和疣蝗配子发生时原癌基因c-kit特异性表达比较研究

应用免疫组织化学和生物统计分析相结合的方法,对花胫绿纹蝗Aiolopus tamulus (Fabricius)和疣蝗Trilophidia annulata (Thunberg)配子发生过程中原癌基因c-kit特异表达进行了比较研究。结果表明：（1）精子发生过程中,精原细胞、初级精母细胞、次级精母细胞和成熟精子中均有不同程度的c-kit蛋白表达,精巢末端还有较粗大的阳性颗粒分布;（2）卵子发生过程中,第1～6阶段卵母细胞中有不同程度的c-kit蛋白特异性表达,但随着卵黄发生的开始逐渐消失;（3）2种蝗虫配子发生过程中c-kit表达存在种间差异。     

猪JARID1C基因的cDNA克隆、生物信息学及组织表达谱分析

JARID1C是高度保守的ARID蛋白家族的成员,该家族的蛋白参与并引起一系列生物学效应,如染色质重塑、细胞增殖与分裂、个体发育以及基因转录调控。JARID1C在人脑中表达丰富,对脑的发育和维持正常功能具有重要作用,突变可引起智力迟钝。本研究采用电子克隆（insilicocloning）的方法并结合5′末端快速扩增技术（RACE）,从猪卵巢中克隆到JARID1C的全长cDNA序列（GenBank登录号：EF139241）。猪JARID1C基因的cDNA全长5,908bp,包括4,551bp的开放阅读框（ORF）、522bp的5′非翻译区（5′UTR）和835bp的3′非翻译区（3′UTR）,polyA加尾信号序列AATAAA位于5,881bp和5,886bp之间。生物信息学分析揭示JARID1C蛋白含有1517个氨基酸残基,定位于细胞核中,该蛋白含有5个保守的结构域：JmjN结构域、ARID结构域、JmjC结构域、C5HC2锌指结构域和PHD锌指结构域。应用ClusterW程序分别对猪、狗、小鼠、大鼠、人和猿的JARID1C核苷酸序列和氨基酸序列进行多重序列比对,发现猪的JARID1C与其他哺乳动物具有很高的相似性。借助Mega3.1软件,采用N-J算法构建JARID1亚家族蛋白的系统进化树,揭示不同物种的进化关系。应用实时荧光定量PCR技术分析该基因在不同组织的表达差异,结果表明该基因在各组织均不同程度地表达,其中在肺和骨骼肌表达水平最低,而在脑和性腺表达水平最高。     

A new epidermal growth factor-like domain in the human core protein for the large cartilage-specific proteoglycan. Evidence for alternative splicing of the domain

A series of cDNA clones for the human core protein of the large cartilage-specific proteoglycan was isolated. Nucleotide sequencing of the clones provided over 2 kilobases of new coding sequences for the human protein. Comparison with published data for cDNA clones covering the same region in rat and chick indicated that domain 8, the lectin-like domain, is highly conserved among species. In contrast, domain 7 is poorly conserved among species. Some of the cDNA clones also contained an additional structural domain between domains 7 and 8 which was not described in the rat or chick sequences. The additional domain of 38 amino acids was highly homologous to epidermal growth factor (EGF)-like sequences seen in other proteins. Because some cDNA clones contained codons for the EGF-like domain and some did not, the results suggested that the EGF-like domain underwent alternative RNA splicing. To confirm alternative splicing of the EGF-like domain, RNA from cartilage cells was used as a template for the polymerase chain reaction. Products of two sizes were obtained. One had the size predicted for mRNA containing the domain and the other had the size predicted for mRNA not containing the domain. Alternative splicing of an EGF-like domain may provide a mechanism of feedback regulation for both the biosynthetic activity and the proliferation of cartilage cells.     

鳜两种生长抑素受体cDNA全长克隆与组织表达

利用cDNA末端快速扩增(RACE)技术克隆了鳜(Siniperca chuatsi)脑中2种生长抑素受体(somatostatin receptor,SSTR2和SSTR3)cDNA全长序列。结果显示,鳜SSTR2 cDNA全长1 820 bp,含开放阅读框1 146 bp,编码382个氨基酸;SSTR3 cDNA全长1 874 bp,含开放阅读框1 458 bp,编码486个氨基酸。SSTR均由5个结构区域组成:N端、7个转膜区(TMD)、3个细胞外袢(ECLs)、4个细胞内袢(ICLs)和C末端。NJ系统进化树分析显示,鳜SSTR2和SSTR3分别形成相对独立的分支,两者间的氨基酸序列相似度为51.2%,表明它们是由不同基因编码而成。利用实时荧光定量RT-PCR技术检测了鳜SSTR2和SSTR3 mRNA的组织表达特征,它们均在多种组织中广泛表达,SSTR2 mRNA在肝中表达量最高,SSTR3 mRNA在胃中表达量最高。SSTR2、SSTR3表达差异反映它们可能参与不同生理调控作用。     

Molecular cloning and in vitro properties of the recombinant rabbit secretin receptor

The rabbit secretin receptor cDNA was cloned from rabbit pancreas using combined polymerase chain reaction (PCR)/rapid amplification of cDNA ends (PCR/RACE) approaches. The rabbit cDNA encoded 445 amino acids and had 80 and 85% homology with rat- and human receptor, respectively, in terms of nucleic and amino acid sequences. Several regions where the rabbit receptor sequence diverged from the rat/human receptor sequences were observed in the putative extracellular domains of the receptor. A cDNA coding for a similar sequence with a 76 bp deletion after the 5th transmembrane domain was also found; it probably encoded an inactive protein. The whole rabbit secretin receptor cDNA was subcloned in expression vector pCR3.1, then stably and transiently transfected in Chinese hamster ovary (CHO) cells. The pharmacological properties of the rat and rabbit secretin receptor studies were compared by radiolabeled secretin binding, binding inhibition, and adenylate cyclase activation (using secretin analogs and fragments). Porcine secretin was equipotent with rabbit secretin on the rabbit secretin receptor, but fivefold more potent than rabbit secretin on the rat receptor. This was due to the serine → arginine residue replacement, in position 16 of rabbit secretin. Amino terminal modified secretin analogs (secretin(2–27), [E³]secretin, [N³]secretin) and VIP were less potent than secretin on both secretin receptors, but more potent on the rabbit than on the rat receptor. The carboxy-terminally truncated fragment (1–26) had the same reduced potency on rat and rabbit receptors. Thus, the rabbit secretin receptor had original properties, different from those of the rat receptor.     

The rat interleukin 10 receptor: cloning and sequencing of cDNA coding for the alpha-chain protein sequence, and demonstration by western blotting of expression in the rat brain

cDNA coding for the alpha chain of the rat interleukin 10 (IL-10) receptor was amplified by polymerase chain reaction (PCR), cloned and sequenced. The nucleic acid coding sequence exhibited 88% and 68% homology with the mouse and human IL-10 receptor sequences, respectively. The translated protein exhibited 83% and 61% homology with the mouse and human IL-10 receptor proteins. Specific antibodies were raised to the extracellular domain of the rat IL-10 receptor expressed as a secreted protein in recombinant Drosophila S2 cells. Western blotting using these antibodies demonstrated the presence of the IL-10 receptor in five major regions of the rat brain (cortex, cerebellum, hippocampus, hypothalamus and pituitary), supporting a role for IL-10 as a central regulator of inflammation.     

Cloning and Characterization of the cDNA Encoding a Novel Brain-Specific 14-kDa Protein

A new acidic protein with a molecular weight of 14,000 was purified from rat brain, in which it was specifically expressed, and partially sequenced by protein sequencing. On the basis of results obtained from the amino acid sequences, mixed oligonucleotides were synthesized and used as probes to clone a cDNA from a rat brain cDNA library. The cloned cDNA provided the full-length sequence of the 14-kDa protein. Northern blot hybridization using total RNA from several tissues of the rat provided evidence that the 14-kDa protein was expressed specifically in rat brain. Transfection of this cDNA into mammalian cells resulted in expression of the 14-kDa protein. The amino acid sequence predicted from the cDNA of the rat brain 14-kDa protein contained 137 amino acid residues. A hydropathy profile revealed a hydrophobic domain (amino acids 60-80) flanked by highly hydrophilic stretches on both sides. Whereas the N-terminal region of the 14-kDa protein contained four repeating motifs, EKTKEGV, the C-terminal domain was rich in glutamic acid and proline. A computer search of the amino acid sequence of the 14-kDa protein indicated no homology to any other protein reported so far.     

cDNA sequence analysis of ribosomal protein S13 gene in Plutella xylostella （Lepidoptera： Plutellidae）

Ribosomal protein S 13 gene has been cloned and analyzed in many organisms,but there are few documents relating to insects. In this communication, the full-length cDNA sequence of ribosomal protein S 13 gene in the diamondback moth, Plutella xylostella(Lepidoptera: Plutellidae), was determined by using PCR amplification technique. The features of the ribosomal protein S 13 gene sequence were analyzed and the deduced amino acids sequence was compared with those from other insects. The results of multi-alignment of the amino acid sequences between the diamondback moth and other insect species revealed that this gene sequence is highly conserved in insects. Based on maximum likelihood method, a phylogenetic tree was constructed from 10 different species using PHYLIP software. It showed that nematode is one separate lineage and the five insect speciesbe long to another lineage, whereas those species higher than insects form the third one. The pattern of this phylogenetic tree evidently represented the evolution of different species.     

Receptor tyrosine kinase, an autapomorphic character of metazoa: identification in marine sponges

In the present review we summarize sequence data obtained from cloning of sponge receptor tyrosine kinases [RTK]. The cDNA sequences were mainly obtained from the marine sponge Geodia cydonium. RTKs (i) with immunoglobulin [Ig]-like domains in the extracellular region, (ii) of the type of insulin-like receptors, as well as (iii) RTKs with one extracellular speract domain, have been identified. The analyses revealed that the RTK genes are constructed in blocks [domains], suggesting a blockwise evolution. The phylogenetic relationships of the sequences obtained revealed that all sponge sequences fall into one branch of the evolutionary tree, while related sequences from higher Metazoa, human, mouse and rat, including also invertebrate sequences, together form a second branch. It is concluded that the RTK molecules have evolved in sponges prior to the "Cambrian Explosion" and have contributed to the rapid appearance of the higher metazoan phyla and that sponges are, as a taxon, also monophyletic. Due to the fact that protein tyrosine kinases in general and RTKs in particular have only been identified in Metazoa, they are, as a group qualified, to be considered as an autapomorphic character of all metazoan phyla.