Description and partial characterization of a new chlamydia-like microorganism

Abstract A new obligate intracellular bacterium which we called 'Z' was isolated as a cell culture contaminant of unknown origin. The organism grew in a variety of cultured cells with a 5–7-day developmental cycle, within cytoplasmic phagosomes, similarly to Chlamydia and some Rickettsia spp. Two alternating developmental forms (elementary bodies and reticulate bodies) were observed by electron microscopy. SDS-PAGE electrophoresis, immunoblotting with chlamydia-specific antibodies, and polymerase chain reaction using chlamydial genus specific primers provided evidence that our bacterium differs significantly from chlamydiae. Further characterization of 'Z' including determination of 16S ribosomal RNA sequences will allow its taxonomic position to be established.     

Comparative genomic signature representations of the emerging COVID-19 coronavirus and other coronaviruses: High identity and possible recombination between Bat and Pangolin coronaviruses

Coronaviruses are responsible on respiratory diseases in animal and human. The combination of numerical encoding techniques and digital signal processing methods are becoming increasingly important in handling large genomic data. In this paper, we propose to analyze the SARS-CoV-2 genomic signature using the combination of different nucleotide representations and signal processing tools in the aim to identify its genetic origin. The sequence of SARS-CoV-2 was compared with 21 relevant sequences including Bat, Yak and Pangolin coronavirus sequences. In addition, we developed a new algorithm to locate the nucleotide modifications. The results show that the Bat and Pangolin coronaviruses were the most related to SARS-CoV-2 with 96% and 86% of identity all along the genome. Within the S gene sequence, the Pangolin sequence presents local highest nucleotide identity. Those findings suggest genesis of SARS-Cov-2 through evolution from Bat and Pangolin strains. This study offers new ways to automatically characterize viruses.     

The rat kidney acylase 1. Evidence for a new cDNA form and comparisons with the porcine intestinal enzyme

A new cDNA form encoding the rat kidney acylase I was characterized and found to show as much as 93.5% identity in its translated nucleotide sequence and, to a lesser extent, in its 3'-untranslated region with the nucleotide sequence we previously reported in 2000. Comparisons between the amino acid sequences of the two corresponding proteins showed the presence of N-terminal fragments with 88.5% identity and different cysteine profiles. The cDNA nucleotide sequence of the pig intestinal enzyme isolated from a marathon library turned out to be 100% identical to that of the kidney enzyme, but differed from those of the two rat kidney acylase I forms.     

Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme

The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.     

人溶酶体酸性b-葡萄糖脑苷脂酶基因的克隆及其在COS7细胞中的表达

从人胎盘组织提取总RNA, 采用RT-PCR扩增人溶酶体酸性b-葡萄糖脑苷脂酶(Lysosomal acid b-glucosidase, GlcCerase)基因编码区的全部序列, 并克隆到pMD-19T载体上, 构建克隆载体pMD-GlcCerase。经测序验证后, 将GlcCerase亚克隆至表达载体pEGFP-C1上, 构建了人GlcCerase绿色荧光蛋白真核表达载体pEGFP-GlcCerase。采用脂质体法将其瞬时转染至COS7细胞系后, 在细胞中检测到了GlcCerase基因, 并在细胞裂解产物中检测到了GlcCerase生物活性的表达。GlcCerase基因的克隆及其表达, 为进一步了解GlcCerase基因的功能以及利用转基因动物乳腺生物反应器高效生产GlcCerase奠定了基础。     

Characterization of the ribosomal RNA locus of Perkinsus atlanticus and development of a polymerase chain reaction-based diagnostic assay

The rRNA locus of Perkinsus atlanticus from the clam Ruditapes decussatus cultivated on the Atlantic coast of Spain was cloned and sequenced. Sequences of the internal transcribed spacer (ITS) from the rRNA locus were compared to sequences reported earlier for a P. atlanticus isolate from Portugal and to those from other Perkinsus species. The ITS I sequence of the Spanish P. atlanticus isolate was identical to the Portuguese P. atlanticus sequence and had 76.6% identity to the ITS1 of Perkinsus marinus. The ITS2 sequence had 99.7% identity to the Portuguese P. atlanticus ITS2, 92.5% identity to the P. marinus ITS2, and 99.5% identity to the Perkinsus olseni ITS2. We report for first the time the small subunit (SSU) and nontranscribed spacer (NTS) of P. atlanticus. The P. atlanticus SSU sequence was 99.6% identical to that of an unidentified Perkinsus species from the Australian clam Anadara trapezia and 98.0% identical to that of P. marinus. Further, our results support the proposal that P. atlanticus, P. olseni, and the Perkinsus sp. from A. trapezia constitute a subgroup of Perkinsus species distributed in the Pacific and eastern Atlantic, different from P. marinus that is distributed along the western edge of the Atlantic. Based on the NTS sequence of P. atlanticus from Spain and the differences with P. marinus NTS (62.2% identity), we developed a polymerase chain reaction (PCR)-based diagnostic assay with a lowest limit of detection of 0.01 amol of cloned NTS DNA as assessed on ethidium bromide-stained agarose gels. Specificity of the PCR-based assay was tested with samples from the clams R. decussatus, Ruditapes philippinarum, and Venerupis pullastra collected in P. atlanticus-enzootic areas of Spain. The specificity and sensitivity demonstrated for this NTS-based PCR assay validate its use as a tool for assessment of P. atlanticus in molluscs.     

Draft Genome Sequence of the Marine Actinomycete Streptomyces sulphureus L180, Isolated from Marine Sediment

Marine-derived actinobacteria are rich sources of valuable natural products and industrial enzymes for biotechnology applications. The marine-derived Streptomyces sulphureus strain L180 was isolated from the marine sediment in a sea cucumber farm at a depth of about 10 m in Dalian, China, and its 16S rRNA gene sequence was determined to have the highest identity to that of Streptomyces sulphureus NRRL B-1627(T) (99.65%). Here, we report the draft genome sequence of this strain.     

Purification, characterization and gene analysis of N-acetylglucosaminidase from Enterobacter sp. G-1.

Enterobacter sp. G-1 is a bacterium isolated previously as a chitinase-producing bacterium. We found this bacterium also produced N-acetylglucosaminidase and characterized that in this study. Extracellular N-acetylglucosaminidase of 92.0 kDa was purified near homogeneity by 8.57-fold from Enterobacter sp. G-1. The optimum temperature and the optimum pH of the purified N-acetylglucosaminidase was 45 degrees C and 6.0, respectively. The N-terminal amino acid sequence of 23 residues of N-acetylglucosaminidase was identified. Based on the N-terminal sequence, we amplified pieces of the DNA fragments by PCR. Using these PCR products as probes, we screened the genomic library and successfully isolated the entire N-acetylglucosaminidase gene (designated nag1) from Enterobacter sp. G-1. The nucleotide sequence of the nag1 gene was found to consist of 2,655 bp encoding a protein of 885 amino acid residues. Comparison of the deduced amino acid sequence from the nag1 gene found 97.3% identity with chitobiase from Serratia marcescens, 54.4% identity with N,N'-diacetylchitobiase from Vibrio harveyi, and 42.7% identity with N-acetylglucosaminidase (ExoI) from Vibrio furnissii. Enzymatic activity assay of N-acetylglucosaminidase indicated stronger activity toward PNP-GlcNAc than PNP-(GlcNAc)2 or PNP-(GlcNAc)3.     

Ferritin acts as a target site for the snowdrop lectin (GNA) in the midgut of the cotton leafworm Spodoptera littoralis

The snowdrop lectin GNA (Galanthus nivalis agglutinin) has been shown to possess insecticidal activity to a range of economically important insect pests. However, the precise mechanism of insecticidal action of GNA against insects remains unknown. In this investigation, we attempted to purify and identify receptor(s) responsible for binding of GNA in the larval midgut of a major lepidopteran pest (the cotton leafworm, Spodoptera littoralis) to better understand its mode of action. Therefore, cytoplasmic as well as membrane proteins from 800 larval midguts were chromatographed on a column with immobilized GNA. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis of the proteins eluted from the GNA column followed by sequencing of the GNA‐binding proteins and BLAST analyses revealed that the N‐terminal sequences of a 24 kDa polypeptide purified from the cytoplasmic and membrane protein fraction revealed sequence similarity to sequences encoding heavy chain homologs of ferritin from Manduca sexta (76% sequence identity), Calpodes ethlius (80% sequence identity) and Bombyx mori (61% sequence identity). Furthermore, the N‐terminal sequence of a 31 kDa polypeptide from the membrane protein fraction showed sequence similarity to a light chain homolog of ferritin from Manduca sexta (88% sequence identity).     

白毛黑眼兔眼部虹膜Trp1、Trp2基因克隆与分析

目的 克隆日本大耳白兔白毛黑眼系(白毛黑眼兔)眼部虹膜Trp1、Trp2基因,获取其完整的外显子序列.进一步推测这两个基因编码的蛋白,并分析其特征.方法 从白毛黑眼兔的黑色虹膜组织中提取RNA,并反转录成cDNA.利用来自小鼠、大鼠和人的同源引物,扩增获得白毛黑眼兔Trp1、Trp2基因外显子片段.然后对已知片段进行3' RACE和5'RACE,从而获得白毛黑眼兔Trp1、Trp2基因外显子完整序列.利用相关软件对获得序列进行翻译和分析.结果 首次获得了白毛黑眼兔Trp1、Trp2基因外显子全序列.该实验兔Trp1基因的编码序列全长1604个碱基,其核苷酸序列与人的相似度为87.9％,与小鼠的相似度为82.7％.TRP1成熟蛋白包含513氨基酸,氨基酸序列与人的相似度为89.8％,与小鼠的相似度为86.6％.该实验兔Trp2基因序列全长1554个碱基,其核苷酸序列与人的相似度为83.2％,与小鼠的相似度为81.9％.TRP2成熟蛋白包含494个氨基酸,其序列与人的一致度为84.2％,与小鼠的一致度为84.4％.结论 本研究获得的TRP1、TRP2的序列与已知的家兔酪氨酸相关蛋白家族成员TYR的序列进行比对,结果显示这三种蛋白之间有较高的相似度,并且TRP1和TRP2蛋白序列表现出了酪氨酸酶家族结构上的保守性和特有的结构特征.     

Remarkable sequence conservation of the last intron in the PKD1 gene

The last intron of the PKD1 gene (intron 45) was found to have exceptionally high sequence conservation across four mammalian species: human, mouse, rat, and dog. This conservation did not extend to the comparable intron in pufferfish. Pairwise comparisons for intron 45 showed 91% identity (human vs. dog) to 100% identity (mouse vs. rat) for an average for all four species of 94% identity. In contrast, introns 43 and 44 of the PKD1 gene had average pairwise identities of 57% and 54%, and exons 43, 44, and 45 and the coding region of exon 46 had average pairwise identities of 80%, 84%, 82%, and 80%. Intron 45 is 90 to 95 bp in length, with the major region of sequence divergence being in a central 4-bp to 9-bp variable region. RNA secondary structure analysis of intron 45 predicts a branching stem-loop structure in which the central variable region lies in one loop and the putative branch point sequence lies in another loop, suggesting that the intron adopts a specific stem-loop structure that may be important for its removal. Although intron 45 appears to conform to the class of small, G-triplet-containing introns that are spliced by a mechanism utilizing intron definition, its high sequence conservation may be a reflection of constraints imposed by a unique mechanism that coordinates splicing of this last PKD1 intron with polyadenylation.     

Prediction of Protein Secondary Structure Using Feature Selection and Analysis Approach

The prediction of the secondary structure of a protein from its amino acid sequence is an important step towards the prediction of its three-dimensional structure. However, the accuracy of ab initio secondary structure prediction from sequence is about 80 % currently, which is still far from satisfactory. In this study, we proposed a novel method that uses binomial distribution to optimize tetrapeptide structural words and increment of diversity with quadratic discriminant to perform prediction for protein three-state secondary structure. A benchmark dataset including 2,640 proteins with sequence identity of less than 25 % was used to train and test the proposed method. The results indicate that overall accuracy of 87.8 % was achieved in secondary structure prediction by using ten-fold cross-validation. Moreover, the accuracy of predicted secondary structures ranges from 84 to 89 % at the level of residue. These results suggest that the feature selection technique can detect the optimized tetrapeptide structural words which affect the accuracy of predicted secondary structures.     

Expression of vitellogenin receptor in the ovarian follicles during the reproductive cycle of the spotted ray Torpedo marmorata Risso 1880

The aim of this investigation was to identify the encoding sequence of vitellogenin receptor gene (vtgr), and its expression during the oogenesis in the spotted ray, Torpedo marmorata, in different phases of reproductive cycle. From an ovarian cDNA of vitellogenic female, we obtained a fragment of 581?bp, which corresponds to a partial sequence encoding the vitellogenin receptor (VTGR) in Torpedo (accession number: gi/193244760). This sequence shows a high identity with the VTGR of other vertebrates, particularly Leucoraja erinacea (89% identity) and Squalus acanthias (84% identity). We also showed that vtgr mRNA expression in the ovary modifies during the oogenesis and throughout the reproductive cycle. Indeed, in immature females, whose ovary contains only previtellogenic follicles, vtgr mRNA occurred in the oocyte cortex as well as within intermediate and pyriform cells. In mature females, whose ovary contains pre- and vitellogenic follicles, vtgr mRNA was detectable not only in the oocyte cortex and in intermediate and pyriform cells but also in small follicle cells present in the follicular epithelium of vitellogenic follicles. In ovulating females, that, as pregnant ones, show pre-and vitellogenic follicles, vtgr mRNA was evident in the oocyte cortex only, whereas in pregnant females, no vtgr mRNA was evident. The role of VTGR in the control of Torpedo vitellogenesis is discussed.     

家蚕丝素重链启动子驱动DsRed的瞬时分泌表达

根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,克隆了家蚕丝素重链基因(Fib-H)启动子及其下游的信号肽序列(FibHS),将DsRed基因与信号肽序列融合构建了分泌型瞬时表达载体;转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed;家蚕注射载体后,可在丝腺腔中检测到红色荧光,表明瞬时表达的DsRed分泌到丝腺腔,推测所克隆的序列具有信号肽的功能。此外,本研究为家蚕丝腺生物反应器分泌表达外源基因的研究奠定了基础。     

Identification of Tropomyosins as Major Allergens in Antarctic Krill and Mantis Shrimp and Their Amino Acid Sequence Characteristics

Tropomyosin represents a major allergen of decapod crustaceans such as shrimps and crabs, and its highly conserved amino acid sequence (>90% identity) is a molecular basis of the immunoglobulin E (IgE) cross-reactivity among decapods. At present, however, little information is available about allergens in edible crustaceans other than decapods. In this study, the major allergen in two species of edible crustaceans, Antarctic krill Euphausia superba and mantis shrimp Oratosquilla oratoria that are taxonomically distinct from decapods, was demonstrated to be tropomyosin by IgE-immunoblotting using patient sera. The cross-reactivity of the tropomyosins from both species with decapod tropomyosins was also confirmed by inhibition IgE immunoblotting. Sequences of the tropomyosins from both species were determined by complementary deoxyribonucleic acid cloning. The mantis shrimp tropomyosin has high sequence identity (>90% identity) with decapod tropomyosins, especially with fast-type tropomyosins. On the other hand, the Antarctic krill tropomyosin is characterized by diverse alterations in region 13–42, the amino acid sequence of which is highly conserved for decapod tropomyosins, and hence, it shares somewhat lower sequence identity (82.4–89.8% identity) with decapod tropomyosins than the mantis shrimp tropomyosin. Quantification by enzyme-linked immunosorbent assay revealed that Antarctic krill contains tropomyosin at almost the same level as decapods, suggesting that its allergenicity is equivalent to decapods. However, mantis shrimp was assumed to be substantially not allergenic because of the extremely low content of tropomyosin.     

Identification by RNA-based arbitrarily primed PCR of the involvement of cytochrome c oxidase in the development of resistance to methotrexate

RNA-based arbitrarily primed PCR (RAP-PCR) was used to identify sequences in CHO K1 cells that were differentially expressed upon methotrexate incubation during the development of resistance to this drug. Ten different RAP products were isolated, cloned and sequenced. Among these, we identified one sequence that showed 84% identity with the nucleotide sequence of rat cytochrome c oxidase subunit II, and 90% identity with the amino acid sequence of this protein. This RAP fragment was up-regulated in a dose- and time-dependent manner. The overexpression of cytochrome c oxidase subunit II mRNA as a result of methotrexate incubation was corroborated by quantitative RT-PCR and Northern blot analysis. Incubation of cells with sodium azide, a specific cytochrome c oxidase inhibitor, decreased the number of resistant colonies after methotrexate treatment. Thus, overexpression of cytochrome c oxidase is involved in the development of resistance to methotrexate. These results suggest that sodium azide may be used as a modulator in chemotherapy with methotrexate.