Nucleotide sequence of the R.meliloti nitrogenase reductase (nifH) gene.

The nucleotide sequence of the structural gene (nifH) of nitrogenase reductase (Fe protein) from R.meliloti 41 with its flanking ends is reported. The amino acid sequence of nitrogenase reductase was deduced from the DNA sequence. The predicted R.meliloti nitrogenase reductase protein consists of 297 amino acid residues, has a molecular weight of 32,740 daltons and contains 5 cysteine residues. The codon usage in the nifH gene is presented. In the 5' flanking region, sequences resembling to consensus sequences of bacterial control regions were found. Comparison of the R.meliloti nifH nucleotide and amino acid sequences with those from different nitrogen-fixing organisms showed that the amino acid sequences are more conserved than the nucleotide sequences. This structural conservation of nitrogenase reductase may be related to its function and may explain the conservation of the nifH gene during evolution.     

Cloning and characterization of nanos gene in silkworm Bombyx mori

Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5＇-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mor/and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5.38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee （Apis mellifera）. This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.     

Expression of the Aspergillus fumigatus rheb homologue,rhbA, is induced by nitrogen starvation

A gene encoding a ras protein with homology to the rheb family was cloned from Aspergillus fumigatus. Although conserved ras domains are present, the predicted RhbA protein sequence deviates from the ras consensus in a manner that is characteristic of rheb proteins. The invariant Gly-Gly in the first GTP-binding domain of ras proteins is replaced by Arg-Ser in RhbA, and a conserved Asp in the effector region of ras proteins is replaced by Asn in RhbA. The rhbA mRNA was detected throughout the A. fumigatus asexual developmental cycle, and accumulated over 5-fold in response to nitrogen starvation. The rhbA gene was able to complement the canavanine hypersensitivity of Saccharomyces cerevisiae Deltarhb1 mutants, suggesting that the two proteins share overlapping function.     

Isolation of a ubiquitin-like (UBL5) gene from a screen identifying highly expressed and conserved iris genes

We have screened a human adult iris cDNA library to identify genes that are highly expressed and conserved between humans and pigs. We identified human iris cDNAs that hybridized at high stringency to a porcine choroidal ring cDNA probe. Of 1568 human iris cDNAs examined, 176 were found to have high expression in porcine choroidal rings. One of the 176 clones was identified as a previously uncharacterized cDNA that we have named the Ubiquitin-like 5 gene (UBL5). The UBL5 gene is located on chromosome 19p13.2, and its genomic structure has been examined. There is a UBL5 pseudogene on chromosome 17p11.2. We have also found homologues to the UBL5 gene in Arabidopsis thaliana, Caenorhabditis elegans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Northern blot analysis of the Ubiquitin-like gene 5 revealed expression in every tissue tested, with the highest levels of RNA expression in heart, skeletal muscle, kidney, liver, iris, and lymphoblasts. Intracellular localization experiments in COS-7 cells showed that the recombinant UBL5 protein is cytoplasmic. Western analysis demonstrated that the recombinant UBL5 protein is approximately 9 kDa, as predicted from the cDNA. A comparison between UBL5 and its homologues with other Ubiquitin-like proteins and Ubiquitin, using the PROTDIST program, suggests that the UBL5 genes are a separate class of Ubiquitin-like genes. Further characterization of the UBL5 gene will determine the function of the encoded protein and whether it is a candidate for ocular disease.     

Human and mouse LSP1 genes code for highly conserved phosphoproteins

With use of the mouse LSP1 cDNA we isolated a human homologue of the mouse LSP1 gene from a human CTL cDNA library. The predicted protein sequence of human LSP1 is compared with the predicted mouse LSP1 protein sequence and regions of homology are identified in order to predict structural features of the LSP1 protein that might be important for its function. Both the human and mouse LSP1 proteins consist of two domains, an N-terminal acidic domain and a C-terminal basic domain. The C-terminal domains of the mouse and human LSP1 proteins are highly conserved and include several conserved, putative serine/threonine phosphorylation sites. Immunoprecipitation of LSP1 protein from 32P-orthophosphate-loaded cells show that both the mouse and human LSP1 proteins are phosphoproteins. The sequences of the putative Ca2(+)-binding sites present in the N-terminal domain of the mouse LSP1 protein are not conserved in the human LSP1 protein; however, a different Ca2(+)-binding site may exist in the human protein, indicating a functional conservation rather than a strict sequence conservation of the two proteins. The expression of the human LSP1 gene follows the same pattern as the expression of the mouse LSP1 gene. Southern analysis of human genomic DNA shows multiple LSP1-related fragments of varying intensity in contrast to the simple pattern found after similar analysis of mouse genomic DNA. By using different parts of the human LSP1 cDNA as a probe, we show that most of these multiple bands contain sequences homologous to the conserved C-terminal region of the LSP1 cDNA. This suggests that there are several LSP1-related genes present in the human genome.     

Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions.

We have isolated and characterized genomic DNA clones for the human and chicken homologues of the mouse En-1 and En-2 genes and determined the genomic structure and predicted protein sequences of both En genes in all three species. Comparison of these vertebrate En sequences with the Xenopus En-2 [Hemmati-Brivanlou et al., 1991) and invertebrate engrailed-like genes showed that the two previously identified highly conserved regions within the En protein ]reviewed in Joyner and Hanks, 1991] can be divided into five distinct subregions, designated EH1 to EH5. Sequences 5' and 3' to the predicted coding regions of the vertebrate En genes were also analyzed in an attempt to identify cis-acting DNA sequences important for the regulation of En gene expression. Considerable sequence similarity was found between the mouse and human homologues both within the putative 5' and 3' untranslated as well as 5' flanking regions. Between the mouse and Xenopus En-2 genes, shorter stretches of sequence similarity were found within the 3' untranslated region. The 5' untranslated regions of the mouse, chicken and Xenopus En-2 genes, however, showed no similarly conserved stretches. In a preliminary analysis of the expression pattern of the human En genes, En-2 protein and RNA were detected in the embryonic and adult cerebellum respectively and not in other tissues tested. These patterns are analogous to those seen in other vertebrates. Taken together these results further strengthen the suggestion that En gene function and regulation has been conserved throughout vertebrate evolution and, along with the five highly conserved regions within the En protein, raise an interesting question about the presence of conserved genetic pathways.     

葡萄风信子MaHY5基因的克隆与表达分析

HY5(ELONGATED HYPOCOTYL5)是促进植物光形态建成过程中非常重要的转录因子。该研究以亚美尼亚葡萄风信子(Muscari armeniacum)为材料,克隆出1个bZIP型转录因子基因MaHY5(GenBank 登录号MK281355)。生物信息学分析显示,MaHY5基因开放阅读框为438 bp,编码145个氨基酸,蛋白质分子量约为16.1 kD,理论等电点为9.78,含有一个保守的bZIP特征结构域;MaHY5蛋白属于亲水性蛋白,无信号肽结构,属于非分泌蛋白,可能定位于细胞核。序列比对和进化树分析表明,HY5在进化过程中高度保守。实时荧光定量PCR和高效液相色谱检测分析表明,MaHY5基因的表达与花青素积累模式基本协同,都在花中优势表达,且在完全开放的花朵中表达较高。 推测MaHY5基因可能参与了葡萄风信子花色素积累过程。     

Molecular Cloning and Identification of the 2′–5′ Oligoadenylate Synthetase 2 Gene in Chinese Domestic Pigs Through Bioinformatics Analysis,and Determination of Its Antiviral Activity Against Porcine Reproductive and Respiratory Syndrome Virus Infection

An interferon-mediated antiviral protein, 2′–5′ oligoadenylate synthetase 2, plays an important role in the antiviral response of interferons. In this study, 2′–5′ oligoadenylate synthetase 2 genes were cloned from Chinese domestic pigs. Bioinformatics analysis revealed that the 2024-bp long open reading fame encodes 707 amino acids. There are two conserved regions in this protein: the nucleotidyltransferase domain, and the 2′–5′ oligoadenylate synthetase domain (OAS). Genetic evolution analysis showed that the 2′–5′ oligoadenylate synthetase 2 gene in domestic pigs is closely related to that of cattle. There are multiple antigenic sites, no signal peptide, and no transmembrane region in the gene, which is predicted to be a hydrophilic protein. Secondary structures were found to be mainly alpha helix-based; its tertiary structure is close to that of humans and cattle, but not that of mice. Tissue distribution results indicated that this protein is distributed in multiple organs, with high distribution in the liver; it is mainly localized in the cytoplasm. PRRSV infection, interferon-beta, and Poly(I: C) treatment all promoted 2′–5′ oligoadenylate synthetase 2 gene expression. Overexpression and RNA silencing of porcine OAS2 inhibited and promoted PRRSV replication in cells, respectively. The inhibitory effect of porcine OAS2 was mainly dependent on RNase L, similar to what was predicted. This study has laid the foundation for future antiviral studies in pig, and provided a new way of preventing and treating PRRSV in the future.     

dfh is a Drosophila homolog of the Friedreich's ataxia disease gene

A putative Drosophila homolog of the Friedreich's ataxia disease gene (FRDA) has been cloned and characterized; it has been named Drosophila frataxin homolog (dfh). It is located at 8C/D position on X chromosome and is spread over 1kb, a much smaller genomic region than the human gene. Its genomic organization is simple, with a single intron dividing the coding region into two exons. The predicted encoded product has 190 amino acids, being considered a frataxin-like protein on the basis of the sequence and secondary structure conservation when compared with human frataxin and related proteins from other eukaryotes. The closest match between the Drosophila and the human proteins involved a stretch of 38 amino acids at C-terminus, encoded by dfh exon 2, and exons 4 and 5a of the FRDA gene, respectively. This highly conserved region is very likely to form a functional domain with a beta sheet structure flanked by alpha-helices where the sequence is less conserved. A signal peptide for mitochondrial import has also been predicted in the Drosophila frataxin-like protein, suggesting its mitochondrial localization, as occurs for human frataxin and other frataxin-like proteins described in eukaryotes. The Drosophila gene is expressed throughout the development of this organism, with a peak of expression in 6-12h embryos, and showing a spatial ubiquitous pattern from 4h embryos to the last embryonic stage examined. The isolation of dfh will soon make available specific dfh mutants that help in understanding the pathogenesis of FRDA.     

Bacteriophage T4 gene 44 DNA polymerase accessory protein. Sequences of gene 44 and its protein product

Bacteriophage T4 gene 44 protein is a DNA polymerase accessory protein which is required for T4 DNA replication. We have isolated the gene for 44 protein from a previously constructed lambda-T4 hybrid phage (Wilson, G. G., Tanyashin, V. I., and Murray, N. E. (1977) Mol. Gen. Genet. 156, 203-214). We report here the nucleotide sequence of gene 44 and about 60 nucleotides 5' upstream from its coding region, which is immediately adjacent to gene 45. We have also purified 44 protein from T4-infected cells and submitted it to extensive protein chemistry characterization. Thus, considerable portions of the protein sequence predicted from the DNA sequence were confirmed by direct protein sequencing of peptides or by matching amino acid compositions of purified peptides. A total of 84% of the predicted amino acids was confirmed by the protein data. These studies indicate that gene 44 codes for a polypeptide containing 319 amino acids, with a calculated Mr = 35,371. The coding region of gene 44 is preceded by a potential regulatory region containing sequences homologous to the Escherichia coli (-10) RNA polymerase binding region and to a conserved sequence at -25 to -30 found in other T4 middle genes. In addition, there are sequence similarities in the translation initiation regions of genes 44, 45, and rIIB, all of which are subject to regulation by regA protein.     

Identification of PDZ5, a candidate universal minicircle sequence binding protein of Trypanosoma cruzi

The dodecamer universal minicircle sequence is a conserved sequence present in minicircles of trypanosomatid kinetoplast DNA studied so far. This sequence is recognised by a protein named universal minicircle sequence binding protein, described for Crithidia fasciculata, involved in minicircle DNA replication. We have identified a Trypanosoma cruzi gene homologue of the Crithidia fasciculata universal minicircle sequence binding protein. Similar to the Crithidia fasciculata universal minicircle sequence binding protein, the Trypanosoma cruzi protein, named PDZ5, contains five zinc finger motifs. Pulsed field gel electrophoresis indicated that the pdz5 gene is located in the chromosomal band XX of the Trypanosoma cruzi genome. The predicted amino acid sequence of PDZ5 shows a high degree of similarity with several trypanosomatid zinc finger proteins. Specific antibody raised against Crithidia fasciculata universal minicircle sequence binding protein recognises both the recombinant and endogenous PDZ5. The complete pdz5 coding sequence cloned in bacteria expresses a recombinant PDZ5 protein that binds specifically to the universal minicircle sequence dodecamer. These data strongly suggest that PDZ5 represents a Trypanosoma cruzi universal minicircle sequence binding protein.