Isolation and characterization of mosquito ferritin and cloning of a cDNA that encodes one subunit

Ferritin, an iron storage protein, was isolated from larvae and pupae of Aedes aegypti grown in an iron-rich medium. Mosquito ferritin is a high molecular weight protein composed of several different, relatively small, subunits. Subunits of molecular mass 24, 26, and 28 kDa are equally abundant, while that of 30 kDa is present only in small amounts. The N-terminal sequence of the 24 and 26 kDa subunits are identical for the first 30 amino acids, while that of the 28 kDa subunit differs. Studies using antiserum raised against a subunit mixture showed that the ferritin subunits were present in larvae, pupae, and adult females, and were increased in animals exposed to excess iron. The antiserum also was used to screen a cDNA library from unfed adult female mosquitoes. Nine clones were obtained that differed only in a 27 bp insertion in the 3′ end. Rapid amplification of cDNA ends (RACE) was used to obtain the complete protein coding sequence. A putative iron-responsive element (IRE) is present in the 5′-untranslated region. The deduced amino acid sequence shows a typical leader sequence, consistent with the fact that most insect ferritins are secreted, rather than cytoplasmic proteins. The sequence encodes a mature polypeptide of 20,566 molecular weight, smaller than the estimated size of any of the subunits. However, the sequence exactly matches the N-terminal sequences of the 24 and 26 kDa subunits as determined by Edman degradation. Of the known ferritin sequences, that of the mosquito is most similar to that of somatic cells of a snail. © 1995 Wiley-Liss, Inc.     

Cloning, expression and tissue distribution of the gene encoding rat fibroblast growth factor receptor subtype 4

The rat homologue of the gene encoding the fibroblast growth factor receptor subtype 4 (FGFR4) was cloned from rat lung mRNA, and the cDNA sequence was found to be 95% similar and 92% identical to the human homologue. Northern blot analysis of adult rat tissues demonstrated that a 3.1-kb mRNA encoding FGFR4 is detectable only in the lung and kidney. The receptor variant described here encodes two potential immunoglobulin-like domains, 21 hydrophobic amino acids encoding a potential transmembrane domain, and a split tyrosine kinase motif. However, the acidic box and hydrophobic signal peptide domains are not present in this cDNA isolate.     

Isolation and characterization of a rice cDNA which encodes a ubiquitin protein and a 52 amino acid extension protein

We isolated a rice cDNA clone encoding the ubiquitin protein fused to a ribosomal protein. This clone encodes a single ubiquitin polypeptide and extension protein of 53 amino acids. This extension protein shows a high degree of homology with those of the yeast ubil or ubi2 gene, both of which encode the same protein. Northern blot analysis suggested that the expression pattern of this gene is more similar to other ribosomal protein genes not linked to ubiquitin protein than to the polyubiquitin gene.     

Isolation and characterization of a human interleukin 2 gene

An interleukin 2 (IL-2) gene was isolated from a Charon 4A human gene library. Electron microscopic examination of 15 heteroduplexes formed between the genomic DNAs and the IL-2 cDNAs demonstrated that the size of the IL-2 gene is about 5.1 +/- 0.5 kb and that there are at least two introns in this gene. Nucleotide sequence of the 5' flanking region of the IL-2 gene showed a homology with that of the corresponding region of the human immune interferon gene.     

Cloning and characterization of DPPL1 and DPPL2, representatives of a novel type of mammalian phosphatidate phosphatase

Phosphatidate phosphatase (PAP) enzymes are classified as either Mg(2+)-dependent (PAP1) or Mg(2+)-independent (PAP2) with respect to their Mg(2+) cofactor requirement for catalytic activity. Sensitivity to the thioreactive compound N-ethylmaleimide (NEM) has also been used to differentiate PAP1 (NEM-sensitive) from PAP2 (NEM-insensitive) activity in mammalian cells. We report here the cloning and initial characterization of DPPL1 and DPPL2, representatives of a novel type of mammalian phosphatidate phosphatase. Both DPPL1 and DPPL2 show greater homology to a yeast diacylglycerol pyrophosphate (DGPP) phosphatase, DPP1, than to known phosphatidate phosphatases of mammals. Like the yeast DPP1 protein, both DPPL1 and DPPL2 proteins show broad substrate specificity, but DGPP is the preferred substrate compared with LPA and PA. These reactions are Mg(2+)-independent, but unlike DPP1 and mammalian PAP2, they are sensitive to NEM. DPPL1 mRNA is ubiquitously expressed in various tissues and cells, but DPPL2 mRNA is restricted to several tissues including the brain, kidney and testis, and it is preferentially expressed in endothelial cells. Immunohistological staining of synovium containing vessels, plasma cells and lymphocytes revealed specific expression of DPPL2 protein in the endothelium. Collectively, our work indicates that DPPL1 and DPPL2 represent a novel type of mammalian phosphatidate phosphatase.     

Isolation and characterization of a new gene, sre, which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa

Multiple GATA factors – regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA core sequence – exist in the filamentous fungus Neurospora crassa. One GATA factor, NIT2, controls nitrogen metabolism, whereas two others, WC-1 and WC-2, regulate genes responsive to blue light induction. A gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method. Sequence analysis of the new GATA factor gene revealed an ORF specifying 587 amino acids, which is interrupted by two small introns. Unlike all previously known Neurospora GATA factors, which possess a single zinc-finger DNA-binding motif, SRE contains two GATA-type zinc fingers. The deduced amino acid sequence of SRE shows significant similarity to URBS1 of Ustilago and SREP of Penicillium. A loss-of-function mutation was created by the RIP procedure. Analysis of sre ⁺ and sre ^? strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores. Siderophore biosynthesis is repressed by high iron concentrations in the wild-type strain but not in sre ^? mutant cells. The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells.     

粗毛栓菌诱变菌株SAH-12漆酶基因的克隆与序列分析

粗毛栓菌Trametes gallica诱变菌株SAH-12是通过紫外诱变选育得到的漆酶高产菌株。为了对其漆酶基因进行研究和利用,采用cDNA末端快速扩增(Rapid Amplification of cDNA Ends,RACE)技术,从T.gallica诱变菌株SAH-12分离得到漆酶基因全长cDNA Lacc1(GenBank accession No.DQ431716)及其对应的结构基因Lac1(DQ431715)。该基因属于真菌漆酶基因家族,与来自出发菌T.gallica漆酶基因lacA(AY875867)在成熟肽编码区的同源性最高(一致性为98%)。Lacc1全长1891bp,由40bp的5'-UTR、1554bp的完整ORF和297bp的3'-UTR构成,具有polyA加尾信号AATACA和59bp的polyA结构;其完整ORF可编码21个氨基酸残基组成的信号肽和496个氨基酸残基组成的成熟蛋白。在Lacc1基因的推导氨基酸序列中有4个潜在的N-糖基化位点和4个参与二硫键形成的Cys残基,且含有真菌漆酶Ⅰ、Ⅱ、Ⅲ型铜离子结合区的4个高度保守序列。结构基因Lac1全长2338bp,含10个内含子和11个外显子,各内含子长度在51bp～76bp之间,且其序列均符合5'-gt……ag-3'规则。     

Two mRNAs exist for the Hs PROS-30 gene encoding a component of human prosomes.

Screening of a λgt11 cDNA expression library of the HeLa cell genome with a monoclonal antibody that specifically recognizes prosomal 30-33-kDa proteins, allowed isolation of a 1264-nucleotide (nt) recombinant cDNA containing a 327-nt untranslated 5'-end. The amino acid (aa) sequence deduced from this cDNA revealed a protein of 269 aa (M_r of 30 227) that includes a consensus box characteristic for Tyr phosphorylation, also observed in other prosomal proteins. Comparison with another prosomal 27-kDa protein, cloned in our laboratory, indicated the presence of three prosomespecific homology boxes observed in these proteins from archaebacteria to man. Interestingly, except for the untranslated 5'-end, as well as the sequence coding for the N-terminal six aa, this cDNA is identical to two recently published cDNAs encoding subunit C2 of human liver proteasome [Tamura et al., Biochim. Biophys. Acta 1089 (1991) 95–102] and subunit NU of human erythrocyte macropain [DeMartino et al., Biochim. Biophys. Acta 1079 (1991) 29–38]. Primer extension and Northern blot analysis using two specific 18-mer oligodeoxyribonucleotides indicated the presence of two mRNAs that have divergent 5'-ends. These results, as confirmed by the polymerase chain reaction, establish the existence of two distinct Hs PROS-30 mRNAs, differing in their 5'-noncoding regions and in the N-terminal six aa of their protein products.     

A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens diseases at the molecular level. ζ-crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several ζ-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3′ end of the coding region. The deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic ζ-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the α-crystallin gene disclosed a dinucleotide delection of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered ζ-crystallin protein. This if the first time a genomic mutation in an enzyme/crytallin gene has been directly linked to a congenital cataract.     

HGV广东株和香港株NS5区部分基因的克隆及序列分析

采用HGV NS5特异的2对引物,对两个香港株和一个广东株HGV-RNA进行逆转录套式PCR扩增,PCR产物克隆入pUC19,重组质粒转化DH5α和JM109菌株.PCR和酶切法鉴定阳性克隆,双脱氧链末端终止法测定核苷酸序列并进行同源性分析.结果发现核苷酸变异呈散在分布,三株间核苷酸和氨基酸序列同源性分别为93.3%～94%及97%～99.2%,与已报道的中国株(CN)相比,则同源性分别为90%～91.2%和94%～96.3%,与美国株(PNF2161及R10291)相比,为87.1%～89.5%和95.2%～97%,而与西非株(GBV-C)相比,则达91.4%～93.8%和97%～97.9%.提示HGV NS5区核苷酸和氨基酸序列相对保守,不同HGV株存在一定的地区差异.     

粗毛栓菌诱变菌株SAH-12漆酶基因的克隆与序列分析

粗毛栓菌Trametes gallica诱变菌株SAH-12是通过紫外诱变选育得到的漆酶高产菌株。为了对其漆酶基因进行研究和利用,采用cDNA末端快速扩增(Rapid Amplification of cDNA Ends,RACE)技术,从T.gallica诱变菌株SAH-12分离得到漆酶基因全长cDNA Lacc1(GenBank accession No.DQ431716)及其对应的结构基因Lac1(DQ431715)。该基因属于真菌漆酶基因家族,与来自出发菌T.gallica漆酶基因lacA(AY875867)在成熟肽编码区的同源性最高(一致性为98%)。Lacc1全长1891bp,由40bp的5'-UTR、1554bp的完整ORF和297bp的3'-UTR构成,具有polyA加尾信号AATACA和59bp的polyA结构;其完整ORF可编码21个氨基酸残基组成的信号肽和496个氨基酸残基组成的成熟蛋白。在Lacc1基因的推导氨基酸序列中有4个潜在的N-糖基化位点和4个参与二硫键形成的Cys残基,且含有真菌漆酶Ⅰ、Ⅱ、Ⅲ型铜离子结合区的4个高度保守序列。结构基因Lac1全长2338bp,含10个内含子和11个外显子,各内含子长度在51bp~76bp之间,且其序列均符合5'-gt…ag-3'规则。     

粗毛栓菌诱变菌株SAH-12漆酶基因的克隆与序列分析

粗毛栓菌Trametes gallica诱变菌株SAH-12是通过紫外诱变选育得到的漆酶高产菌株。为了对其漆酶基因进行研究和利用,采用cDNA末端快速扩增(Rapid Amplification of cDNA Ends,RACE)技术,从T.gallica诱变菌株SAH-12分离得到漆酶基因全长cDNA Lacc1(GenBank accession No.DQ431716)及其对应的结构基因Lac1(DQ431715)。该基因属于真菌漆酶基因家族,与来自出发菌T.gallica漆酶基因lacA(AY875867)在成熟肽编码区的同源性最高(一致性为98%)。Lacc1全长1891bp,由40bp的5'-UTR、1554bp的完整ORF和297bp的3'-UTR构成,具有polyA加尾信号AATACA和59bp的polyA结构;其完整ORF可编码21个氨基酸残基组成的信号肽和496个氨基酸残基组成的成熟蛋白。在Lacc1基因的推导氨基酸序列中有4个潜在的N-糖基化位点和4个参与二硫键形成的Cys残基,且含有真菌漆酶Ⅰ、Ⅱ、Ⅲ型铜离子结合区的4个高度保守序列。结构基因Lac1全长2338bp,含10个内含子和11个外显子,各内含子长度在51bp~76bp之间,且其序列均符合5'-gt…ag-3'规则。