Cloning and nucleotide sequence of the murine interleukin-3 gene

Southern hybridization analysis using a probe derived from a murine interleukin-3 (IL-3) cDNA clone revealed the presence of a single IL-3 gene in the haploid murine genome. An 8600-base-pair (8.6-kb) murine genomic EcoRI fragment containing the IL-3 gene was isolated by screening a library of size-fractionated genomic EcoRI fragments cloned in lambda gtWES X lambda B. The nucleotide sequence of a 3.5-kb region of the cloned DNA encompassing the IL-3 gene was determined. The gene contains four introns of 96, 993, 135 and 122 base pairs (bp), located within the coding region. The large intron contains 12 copies of a 14-15-bp tandem repeating sequence which resembles a human cellular homologue of a BKV enhancer sequence. The nucleotide sequence of the exons agrees exactly with that of an IL-3 cDNA cloned from WEHI-3, a tumorigenic cell line which over-produces IL-3, establishing that the unprocessed primary structure of IL-3 is identical in WEHI-3 and in BALB/c mice. Southern hybridization has revealed genomic alteration in the vicinity of the IL-3 gene in WEHI-3 cells.     

Molecular cloning of a human immunoglobulin lambda chain variable sequence

We have cloned a human V lambda cDNA sequence from an Ig lambda-producing human Burkitt lymphoma cell line (BL2) by taking advantage of a cloned constant region gene as a primer for cDNA synthesis instead of an oligo(dT) primer. The amino acid sequence deduced from the nucleotide sequence of V lambda clones is highly related to that of the NEW V lambda protein of subgroup I. Southern blot hybridization of human DNAs with the V lambda I probe showed at least 12 hybridizing V lambda fragments. These fragments are amplified in K562 cells which derive from a case of chronic myelogenous leukemia and contain an amplified c-abl oncogene and amplified C lambda sequences.     

Nucleotide sequence of mouse IL-2 receptor cDNA and its comparison with the human IL-2 receptor sequence.

We have cloned cDNA encoding the mouse interleukin-2 (IL-2) receptor from a murine T cell line, CTLL using human IL-2 receptor cDNA as probe. COS 7 cells transfected with the cDNA expressed the antigen recognized by the monoclonal antibody against the murine IL-2 receptor. The cDNA identified 4 species of mRNA (4.5, 3.5, 2.2 and 1.5 kb) of the mouse IL-2 receptor in CTLL cells. Difference in the length of mRNA seems to be ascribed to the variable length of the 3' untranslated sequence. Total nucleotide sequence (approximately 1400 bp) of this cDNA was determined and compared with the human receptor. The nucleotide and amino acid sequences of the IL-2 receptor are 70% and 60%, respectively, homologous in average between the two species. The comparison has revealed several conserved regions localized to particular exons such as transmembrane and cytoplasmic portions, suggesting that these regions are important for receptor function and its regulation.     

Amplification and direct nucleotide sequencing of cDNA from the lysate of low numbers of diploid human cells

The polymerase chain reaction technique is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. However, some investigators need to sequence the entire coding region of mammalian genes, e.g., cellular ras genes or the gene encoding hypoxanthine (guanine) phosphoribosyl transferase (HPRT), to determine what specific changes have occurred. To do so, they isolate RNA from large populations of cells, amplify cDNA from the gene of interest, subclone the product, and sequence two or more isolates to determine the common mutation. We have developed a method to simplify this procedure by copying mRNA of the hprt gene directly from the lysate of a clone of mutant diploid human fibroblasts (e.g., 100 cells). We amplified the first and second strand of the cDNA of the gene of interest 10(10)- to 10(11)-fold, obtained 5 to 10 micrograms of DNA in less than 10 h, and sequenced the coding region directly without the need for RNA extraction or DNA template purification. By our method cDNA can be amplified directly from the lysate of just one human cell, but to avoid detecting random changes introduced by the polymerase, we lysed approx. 200 cells from a clone, each containing the identical mutation, amplified the cDNA, and determined the consensus sequence by direct nucleotide sequencing.     

Cloning of the human cDNA which can complement the defect of the yeast mannosyltransferase I-deficient mutant alg 1

The assembly of the lipid-linked oligosaccharide, Glc(3)Man(9)GlcNAc(2)-P-P-Dol, occurs on the rough ER membrane in an ordered stepwise manner. The process is highly conserved among eukaryotes. In order to isolate the human mannosyltransferase I (MT-I) gene involved in the process, we used the Saccharomyces cerevisiae MT-I gene ( ALG1 ), which has already been cloned. On searching the EST database with the amino acid sequence of the ALG1 gene product, we detected seven related human EST clones. A human fetal brain cDNA library was screened by PCR using gene-specific primers based on the EST nucleotide sequences and a 430 bp cDNA fragment was amplified. The cDNA library was rescreened with this 430 bp cDNA, and two cDNA clones (HR1-3 and HR1-4) were isolated and sequenced. On a homology search of the EST database with the nucleotide sequence of HR1-3, we detected a novel human EST clone, AA675921 (GenBank accession number). Based on the nucleotide sequences of AA675921 and HR1-4, we designed gene-specific PCR primers, which allowed to amplify a 1.8 kb cDNA from human fetal brain cDNA. This cDNA was cloned and shown to contain an ORF encoding a protein of 464 amino acids. We designated this ORF as Hmat-1. The amino acid sequence deduced from the Hmat-1 gene showed several highly conserved regions shared with the yeast and nematode MT-I sequences. Furthermore, this 1.8 kb cDNA successfully complemented the S. cerevisiae alg1-1 mutation, indicating that the Hmat-1 gene encodes the human MT-I and that the function of this enzyme was conserved between yeast and human.     

恶性疟原虫海南株(FCC1)翻译控制肿瘤蛋白(TCTP)基因的克隆及序列测定

为了获得翻译控制肿瘤蛋白 (TCTP)基因 ,提取恶性疟原虫海南株 (FCC1)总RNA ,直接用总RNA反转录成单链DNA ,再用长距PCR方法扩增出双链cDNA .根据小鼠约氏疟原虫TCTP基因序列设计引物 ,以cDNA为模板合成了恶性疟原虫海南株TCTP基因 .以pMD18 T为载体 ,用大肠杆菌XL1 blue对基因克隆 .测序结果表明 ,此基因序列与小鼠约氏疟原虫TCTP基因序列有 85 %同源性 .由此基因序列推导出的TCTP氨基酸序列 ,与小鼠约氏疟原虫TCTP氨基酸序列有 88%同源性 .进入GenBank国际基因库 (美国 )检索 ,所克隆的基因与基因库中恶性疟原虫基因同源性为 97% ;由所克隆的基因序列推导的TCTP氨基酸序列 ,与国际基因库恶性疟原虫TCTP基因推导的TCTP氨基酸序列仅有 1个氨基酸差异 .恶性疟原虫海南株TCTP基因克隆为进一步研究奠定了基础     

Discovery of new human beta-defensins using a genomics-based approach

Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.     

Cloning and expression of the rat interleukin-3 gene.

Genomic clones carrying the rat interleukin-3 (IL-3) gene have been isolated and the nucleotide sequence of the gene determined. Alignment of this sequence with that of the mouse IL-3 gene has allowed the structure of the rat IL-3 gene to be deduced. The intron-exon boundaries are conserved and extensive nucleotide homology (approx 90%) is present in the 5' flanking region and the portion of the gene coding for the signal peptide. Several proposed regulatory sequences are conserved and an analogous element to the tandem repeat in intron 2 of the mouse gene is also present. The predicted amino acid sequence for mature rat IL-3 shows surprisingly low homology (54%) with its murine counterpart, although all four cysteine residues are conserved. The rat IL-3 gene was expressed in monkey COS-1 cells and colony assays established that rat IL-3 is a multi-lineage haemopoietic growth regulator. There was little cross-reactivity of the respective IL-3 species on mouse and rat bone marrow cells suggesting that rat IL-3, in concert with its receptor, has evolved significantly away from the mouse IL-3/receptor system.     

Expression of the pS2 gene in human gastric cancer cells derived from poorly differentiated adenocarcinoma

NH2-terminal amino acid sequence of the pS2 protein produced and secreted by human gastric cancer cells, MKN-45, was determined to be identical to that of MCF-7 cells. A clone encoding pS2 protein was isolated from the cDNA library constructed from MKN-45 cells. The nucleotide sequence was identical to that of pS2 cDNA previously isolated from human breast cancer cells, MCF-7, except for one nucleotide in the 3' untranslated region. Thus, in this cell line, the pS2 gene product is translated and secreted as in MCF-7 cells. RNA blot hybridization analysis revealed that pS2 gene was expressed well in two (MKN-45 and KATO-III; derived from poorly differentiated adenocarcinoma) but not in three cell lines (MKN-1, MKN-28 and MKN-74; from well differentiated adenocarcinoma), suggesting that expression of the pS2 gene depends on the state of cell differentiation. These results suggest that pS2 is expressed in human gastric cancer cells in an estrogen-independent manner and is possibly associated with the malignant state of cells.     

Molecular cDNA cloning and analysis of the organization and expression of the IL-1beta gene in the Nile tilapia, Oreochromis niloticus

The full-length cDNA sequence of interleukin-1beta (IL-1beta) from the Nile tilapia, Oreochromis niloticus, was determined by using PCR with primers designed from known fish IL-1beta sequences followed by elongation of the 5' and 3' ends using Rapid Amplification of cDNA Ends (RACE). The cDNA contains a 92-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 732 bp that translates into a 243-amino acid molecule, a 341-bp 3' UTR with four cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly(A) tail. The organization of the genomic IL-1beta based on the cDNA sequence appeared to be 4 introns and 5 exons. In comparison with known IL-1beta amino acid sequences, including human, catshark, trout, turbot, carp, sea bream, sea bass and goldfish, the amino acid sequence deduced from the cDNA sequence of Nile tilapia showed different levels of identity ranging from 25.32% to 66.80% and homology ranging from 41.88% to 82.19%. Although the entire cDNA sequence of Nile tilapia IL-1beta showed from 49.45% to 67.05% identity to those of other reported IL-1beta cDNAs, each exon also showed different levels of identity to the corresponding exons of other reported IL-1beta cDNAs. The highest nucleotide sequence identity for exon 1 and exons 2-5 of Nile tilapia IL-1beta was found in the corresponding exons of sea bream and sea bass, respectively. After in vitro stimulation with lipopolysaccharide (LPS), we found an increased level of IL-1beta expression in head kidney cells compared to that of unstimulated cells. However, this difference was no longer apparent after 4 h of stimulation, at which time the levels were similar in stimulated and unstimulated cells. Head kidney cells stimulated in vivo by an intraperitoneal injection of LPS showed a peak level of IL-1beta expression after 1 day and a decreased level after 3 days. At 7 days after stimulation, we were hardly able to detect IL-1beta expression.     

Identification of IL-18RAP mRNA truncated splice variants in human testis and the other human tissues

IL-18 is a pleiotropic cytokine involved in the regulation of both innate and adaptive immunity. It plays a key role in the autoimmune, inflammatory and infectious diseases. IL-18 acts via a receptor complex that closely resembles that of IL-1, consisting of a ligand binding protein, IL-18Ralpha, and an accessory protein, IL-18RAP (IL-18Rbeta). IL-18RAP is essential for IL-18 signal transduction and ligand binding affinity to IL-18Ralpha receptor chain. mRNA of gene coding for IL-18RAP in human testicular tissue and the nucleotide sequence of splice variants was carefully examined. We have found for the first time ever, IL-18RAP mRNA in studied tissue samples of physiological testis. Using the RT-PCR technique, the whole coding sequence of this gene was amplified. An alternative splicing of mRNA for IL-18RAP was then discovered and subsequently confirmed by cDNA sequencing. The putative amino acid content was predicted and a computer modeling was performed. It might be hypothesized that the truncated forms of IL-18RAP can be involved in the complex mechanism of IL-18 activity regulation.     

The gene encoding hypoxanthine-guanine phosphoribosyltransferase as target for mutational analysis: PCR cloning and sequencing of the cDNA from the rat.

In this paper, the cloning and nucleotide sequence of the cDNA of the rat gene coding for hypoxanthine-guanine phosphoribosyltransferase (hprt) is reported. Knowledge of the cDNA sequence is needed, among other reasons, for the molecular analysis of hprt mutations occurring in rat cells, such as skin fibroblasts isolated according to the granuloma pouch assay. The rat hprt cDNA was synthesized and used as a template for in vitro amplification by PCR. For this purpose, oligonucleotide primers were used, the nucleotide sequences of which were based on mouse and hamster hprt cDNA sequences. Sequence analysis of 1146 bp of the amplified rat hprt cDNA showed a single open reading frame of 654 bp, encoding a protein of 218 amino acids. In the predicted rat hprt amino acid sequence, the proposed functional domains for 5'-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide binding in phosphoribosylating enzymes as well as a region near the carboxyl terminal part were highly conserved when compared with amino acid sequences of other mammalian hprt proteins. Analysis of hprt amino acid sequences of 727 independent hprt mutants from human, mouse, hamster and rat cells bearing single amino acid substitutions revealed that a large variety of amino acid changes were located in these highly conserved regions, suggesting that all 3 domains are important for proper catalytic activity. The suitability of the hprt gene as target for mutational analysis is demonstrated by the fact that amino acid changes in at least 151 of the 218 amino acid residues of the hprt protein result in a 6-thioguanine-resistant phenotype.     

Molecular cloning and high-level expression of human polymerase beta cDNA and comparison of the purified recombinant human and rat enzymes

The cDNA encoding the human polymerase beta from HeLa cells was PCR amplified and cloned, and its nucleotide sequence determined. The DNA sequence is identical to the polymerase beta cDNA sequence from Tera-2 cells. Three expression strategies were employed that were designed to maximize translation initiation of the polymerase beta mRNA in Escherichia coli and all yielded a high level of human polymerase beta. The recombinant protein was purified and its properties were compared with those of the recombinant rat enzyme. The domain structure and kinetic parameters (k(cat) and K(m)) were nearly identical. A mouse IgG monoclonal antibody to the rat enzyme (mAb-10S) was approximately 10-fold less reactive with the human enzyme than with the rat enzyme as determined by ELISA.     

金龟子绿僵菌中性海藻糖酶基因的克隆及其表达特性分析

根据中性海藻糖酶NTL基因的同源序列设计引物,PCR扩增出杀蝗专一菌株———金龟子绿僵菌CQMa102NTL基因片段,利用5′_RACE和3′_RACE扩增出NTLcDNA的5′和3′端序列,经拼接得到CQMa102NTL基因cDNA全长。根据其全长cDNA序列,设计引物PCR扩增出CQMa102NTL的完整基因。为了解该基因的上游调控信息,采用PanhandlePolymeraseChainReactionAmplification方法扩增其上游序列。序列分析表明,CQMa102NTL全长DNA3484bp,cDNA全长2385bp,编码737个氨基酸的蛋白,推测蛋白分子量为83.1kD;含有3个内含子,包含一个依赖于cAMP的磷酸化作用位点(RRGS)和一个钙附着位点(DTDGNMQITIED);上游序列含有一个压力反应元件(CCCCT);与金龟子绿僵菌广谱性菌株ME1NTL的核苷酸序列和氨基酸序列分别具有93%和99%同源性,由此确定该序列为金龟子绿僵菌中性海藻糖酶基因序列。Southern杂交表明,NTL基因在CQMa102基因组中为单拷贝。Northern杂交表明,NTL基因转录出约2.5kb的mRNA单带,在液体培养条件下,对数生长前期表达水平最高,对数生长后期降到最低,进入稳定生长期后表达水平又有所提高。金龟子绿僵菌CQMa102中性海藻糖酶基因DNA全长和cDNA全长登录GenBank,登录号分别为:AY557613,AY557612。     

小鼠白细胞介素-33基因的克隆及序列分析

目的:克隆小鼠IL-33基因全长编码区cDNA,并对其进行序列分析。方法:从BALB/c小鼠的脊髓组织中提取总RNA,逆转录为cDNA,用热启动PCR技术,扩增小鼠IL-33基因全长编码区cDNA,经双酶切后,克隆入pcDNA3.1( )载体中,构建真核表达载体pcDNA3.1-mIL-33,然后进行酶切鉴定与序列分析。结果:小鼠IL-33基因的PCR产物和重组载体经凝胶电泳和酶切鉴定、测序分析证实,其序列与GenBank中数据一致。小鼠IL-33基因的全长编码序列为801 bp,编码266个氨基酸。结论:小鼠IL-33基因成功的克隆并构建了其真核表达载体,为进一步进行IL-33的表达与功能研究奠定了基础。     

小鼠白细胞介素33真核表达质粒的构建及表达

克隆小鼠IL-33基因构建其真核表达质粒,并转染COS-7细胞检测其表达。提取C57BL/6小鼠肺组织总RNA,经反转录聚合酶链式反应（RT-PCR）扩增小鼠IL-33基因,酶切后插入pcDNA^TM3.1/myc HisA构建其真核表达质粒pcDNA-3.1-IL-33,重组质粒转染COS-7细胞,RT-PCR和免疫印迹法（western blotting）检测目的基因表达。结果显示,pcDNA3.1-IL-33中插入的片段序列测定结果与小鼠IL-33cDNA序列一致,重组质粒转染COS-7细胞后检测到相应mRNA及蛋白表达。成功克隆了小鼠IL-33基因cDNA,并构建其真核表达质粒。     

可溶性白细胞介素6受体基因在链霉菌的克隆表达研究

以分离提取的ＨｅＬａ细胞总ＲＮＡ为模板,通过ＲＴ－ＰＣＲ反应扩增得到了１０１７ｂｐ的人可溶性白细胞介素６受体（ｓＩＬ－６Ｒ）ｃＤＮＡ片段,将扩增片段克隆到ｐＵＣ１９质粒中进行序列测定。结果证明该片的序列与文献报道的ｓＩＬ－６ＲｃＤＮＡ的序列完全一致,将ｓＩＬ－６ＲｃＤＮＡ与链霉素信号肽ｍｅｌＣｌ的编码序列融合后得到的融合基因ｍｅｌ／ｓＩＬ－６Ｒ克隆到链霉菌质粒ｐＬＪ４５９中,构建成重组表达质粒ｐＬ