生物大分子从头合成和设计的研究进展

生物大分子指生物体内存在的DNA、蛋白质、多糖等物质,其对生物体正常生命活动至关重要.从头合成和设计技术在生物大分子的合成和结构设计上具有自由度高、前体简单等特点,能够按照特定研究目的对生物大分子进行全新设计和高效合成.近年来,从头合成与设计技术在人造基因组合成、新型蛋白质类药物设计、糖缀合物合成等领域已开始受到重视.基于生物大分子从头合成和设计技术,可以定向制备全新设计的DNA或全新的基因表达产物,以及具有识别功能的糖链或糖缀合物,将大大推进诸如细胞因子模拟物、基因治疗递送载体等生物活性物质的开发,为人工生物系统的构建、罕见疾病的治疗等提供新的解决方法.本文就DNA、蛋白质和多糖的从头合成和设计进行了综述,阐述了相关方法及应用,最后概括分析了三者之间的关系.     

Fabp4‐CreER lineage tracing revealstwo distinctive coronary vascular populations

Over the last two decades, genetic lineage tracing has allowed for the elucidation of the cellular origins and fates during both embryogenesis and in pathological settings in adults. Recent lineage tracing studies using Apln‐CreER tool indicated that a large number of post‐natal coronary vessels do not form from pre‐existing vessels. Instead, they form de novo after birth, which represents a coronary vascular population (CVP) distinct from the pre‐existing one. Herein, we present new coronary vasculature lineage tracing results using a novel tool, Fabp4‐CreER. Our results confirm the distinct existence of two unique CVPs. The 1^st CVP, which is labelled by Fabp4‐CreER, arises through angiogenic sprouting of pre‐existing vessels established during early embryogenesis. The 2^nd CVP is not labelled by Fabp4, suggesting that these vessels form de novo, rather than through expansion of the 1^st CVP. These results support the de novo formation of vessels in the post‐natal heart, which has implications for studies in cardiovascular disease and heart regeneration.     

Argonautes team up to silence transposable elements in Arabidopsis

The de novo silencing of transposable elements in plants and animals is mediated in part by RNA‐directed chromatin modification. In flowering plants, AGO4 has been seen as the key argonaute protein in the RNA‐directed DNA methylation pathway that links the plant‐specific RNA polymerase V with the de novo DNA methyltransferase DRM2 (Zhong et al, 2014). Two recent papers in The EMBO Journal strongly implicate a role for the AGO6 protein in the process of de novo silencing.     

OBSERVATIONS ON PURINE METABOLISM IN RAT BRAIN

Both de novo and preformed base or salvage pathways are simultaneously operative in the biosynthesis of purine nucleotides in rat brain per se. A preferential utilization of de novo precursors is demonstrated.     

Algorithms for the de novo sequencing of peptides from tandem mass spectra

Proteomics is the study of proteins, their time- and location-dependent expression profiles, as well as their modifications and interactions. Mass spectrometry is useful to investigate many of the questions asked in proteomics. Database search methods are typically employed to identify proteins from complex mixtures. However, databases are not often available or, despite their availability, some sequences are not readily found therein. To overcome this problem, de novo sequencing can be used to directly assign a peptide sequence to a tandem mass spectrometry spectrum. Many algorithms have been proposed for de novo sequencing and a selection of them are detailed in this article. Although a standard accuracy measure has not been agreed upon in the field, relative algorithm performance is discussed. The current state of the de novo sequencing is assessed thereafter and, finally, examples are used to construct possible future perspectives of the field.     

Viral Proteins Originated De Novo by Overprinting Can Be Identified by Codon Usage: Application to the “Gene Nursery” of Deltaretroviruses

A well-known mechanism through which new protein-coding genes originate is by modification of pre-existing genes, e.g. by duplication or horizontal transfer. In contrast, many viruses generate protein-coding genes de novo, via the overprinting of a new reading frame onto an existing (“ancestral”) frame. This mechanism is thought to play an important role in viral pathogenicity, but has been poorly explored, perhaps because identifying the de novo frames is very challenging. Therefore, a new approach to detect them was needed. We assembled a reference set of overlapping genes for which we could reliably determine the ancestral frames, and found that their codon usage was significantly closer to that of the rest of the viral genome than the codon usage of de novo frames. Based on this observation, we designed a method that allowed the identification of de novo frames based on their codon usage with a very good specificity, but intermediate sensitivity. Using our method, we predicted that the Rex gene of deltaretroviruses has originated de novo by overprinting the Tax gene. Intriguingly, several genes in the same genomic region have also originated de novo and encode proteins that regulate the functions of Tax. Such “gene nurseries” may be common in viral genomes. Finally, our results confirm that the genomic GC content is not the only determinant of codon usage in viruses and suggest that a constraint linked to translation must influence codon usage.     

PHOSPHONATE ANALOGS OF CARBOCYCLIC PHOSPHORIBOSYLAMINE AND CARBOCYCLIC GLYCINAMIDE RIBONUCLEOTIDE

Abstract

Analogs of intermediates in the de novo purine nucleotide biosynthetic pathway were synthesized to study the binding requirements of the corresponding enzymes. Because of the instability of the natural stubstrates, such as phosphoribosylamine, the use of the structurally stable phosphonate moiety and the carbocyclic ribose yields ideal analogs for these studies. In addition, these analogs can act as potential inhibitors of the de novo pathway leading to the design of anticancer agents. Enzyme studies with GAR synthetase and GAR transformylase reveal that the title compounds can act as substrates or inhibitors of the de novo enzymes.     

Analysis of hypermethylation in the RPS element suggests a signal function for short inverted repeats in de novo methylation

A repetitive DNA sequence (RPS) from Petunia hybrida had previously been shown to enhance expression variegation in petunia and tobacco and to carry a hot spot for de novo DNA methylation. Here we show that a strong de novo hypermethylation site is located within a palindromic segment of the RPS and present indirect evidence, based on sequence homologies to other repeat units within the RPS, for the formation of secondary structures at the methylation site in vivo. We demonstrate that the palindromic RPS element, which is moderately to highly repetitive in petunia, does not predominantly localise to constitutive heterochromatin. To test whether the RPS is subject to de novo methylation due to its repetitive nature or to intrinsic signals within the RPS, we integrated the RPS into the genome of Arabidopsis thaliana, a plant lacking homology to the RPS. Our data indicate that the palindromic element also acts as a de novo hypermethylation site in the non-repetitive genomic background of Arabidopsis, strongly suggesting a signal function of the palindromic RPS element.     

Oncogene dependent requirement of fatty acid synthase in hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the most frequent primary tumor of the liver, is an aggressive cancer type with limited treatment options. Cumulating evidence underlines a crucial role of aberrant lipid biosynthesis (a process known as de novo lipogenesis) along carcinogenesis. Previous studies showed that suppression of fatty acid synthase (FASN), the major enzyme responsible for de novo lipogenesis, is highly detrimental for the in vitro growth of HCC cell lines. To assess whether de novo lipogenesis is required for liver carcinogenesis, we have generated various mouse models of liver cancer by stably overexpressing candidate oncogenes in the mouse liver via hydrodynamic gene delivery. We found that overexpression of FASN in the mouse liver is unable to malignantly transform hepatocytes. However, genetic deletion of FASN totally suppresses hepatocarcinogenesis driven by AKT and AKT/c-Met protooncogenes in mice. On the other hand, liver tumor development is completely unaffected by FASN depletion in mice co-expressing β-catenin and c-Met. Our data indicate that tumors might be either addicted to or independent from de novo lipogenesis for their growth depending on the oncogenes involved. Additional investigation is required to unravel the molecular mechanisms whereby some oncogenes render cancer cells resistant to inhibition of de novo lipogenesis.     

Heterologous expression of naturally evolved putative de novo proteins with chaperones

Over the past decade, evidence has accumulated that new protein‐coding genes can emerge de novo from previously non‐coding DNA. Most studies have focused on large scale computational predictions of de novo protein‐coding genes across a wide range of organisms. In contrast, experimental data concerning the folding and function of de novo proteins are scarce. This might be due to difficulties in handling de novo proteins in vitro, as most are short and predicted to be disordered. Here, we propose a guideline for the effective expression of eukaryotic de novo proteins in Escherichia coli. We used 11 sequences from Drosophila melanogaster and 10 from Homo sapiens, that are predicted de novo proteins from former studies, for heterologous expression. The candidate de novo proteins have varying secondary structure and disorder content. Using multiple combinations of purification tags, E. coli expression strains, and chaperone systems, we were able to increase the number of solubly expressed putative de novo proteins from 30% to 62%. Our findings indicate that the best combination for expressing putative de novo proteins in E. coli is a GST‐tag with T7 Express cells and co‐expressed chaperones. We found that, overall, proteins with higher predicted disorder were easier to express.StatementToday, we know that proteins do not only evolve by duplication and divergence of existing proteins but also arise from previously non‐coding DNA. These proteins are called de novo proteins. Their properties are still poorly understood and their experimental analysis faces major obstacles. Here, we aim to present a starting point for soluble expression of de novo proteins with the help of chaperones and thereby enable further characterization.     

Pyrimidine biosynthesis in rat brain

—Studies on the incorporation of [¹⁴C]NaHCO₃ into both orotic acid and RNA in tissue slices reveal the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in the rat brain. A comparison of the rates of incorporation of bicarbonate into orotic acid and RNA in tissue slices of brain and liver indicate the brain to be one-fourth to one-half as active as the liver in the de novo biosynthesis of pyrimidines. The results of this study favor the proposal that the adult rat brain can meet its needs for pyrimidines through de novo synthesis and is not dependent upon salvage activity and an extraneural supply of pyrimidines.     

Nucleotide Metabolism during Pine Pollen Germination

The metabolism of purine- and pyrimidine nucleotides in pine pollen (Pinus mugo) grown in suspension cultures have been examined. In the ungerminated dehydrated pollen, the presence of ATP has been demonstrated. Incubation of the pollen in a germination medium leads to an exhaustion of the ATP pool, which is restored with the onset of oxygen uptake. By labelling pollen cultures with ³²P-orthophosphate, it has been possible to quantitate the nucleotide components of the pollen, and thereby to measure changes in the nucleotide pattern at various growth stages. The most marked changes occur during the initial phase of tube growth when a large increase in the ribonucleoside triphosphate and the sugar nucleotide pools is observed. The contents of ATP and UDP-glucose are further increased if starch synthesis is initiated by the addition of sucrose to the culture medium. In order to determine whether nucleotides in pine pollen are synthesized from de novo pathways or via reutilization pathways, from breakdown products of nucleic acids, pollen was incubated with ¹⁴C-labelled precursors of both the de novo and the reutilization pathways. Incorporation experiments established de novo synthesis of ATP and GTP from glycine, and de novo synthesis of CTP and UTP from orotic acid. The operation of pathways for the utilization of exogenous nucleosides was also demonstrated. While uridine, cytidine and adenosine are incorporated into nucleoside triphosphate to a great extent, only minor incorporation of inosine and guanosine is observed. These reutilization pathways might be of importance for the synthesis of nucleotides during tube growth in situ. Addition of inhibitors of glycolysis and oxidative phosphorylation drastically reduces the level of ribonucleoside triphosphates, indicating a rapid turnover of the nucleotide pool.     

The evolutionary origins of pesticide resistance

Durable crop protection is an essential component of current and future food security. However, the effectiveness of pesticides is threatened by the evolution of resistant pathogens, weeds and insect pests. Pesticides are mostly novel synthetic compounds, and yet target species are often able to evolve resistance soon after a new compound is introduced. Therefore, pesticide resistance provides an interesting case of rapid evolution under strong selective pressures, which can be used to address fundamental questions concerning the evolutionary origins of adaptations to novel conditions. We ask: (i) whether this adaptive potential originates mainly from de novo mutations or from standing variation; (ii) which pre‐existing traits could form the basis of resistance adaptations; and (iii) whether recurrence of resistance mechanisms among species results from interbreeding and horizontal gene transfer or from independent parallel evolution. We compare and contrast the three major pesticide groups: insecticides, herbicides and fungicides. Whilst resistance to these three agrochemical classes is to some extent united by the common evolutionary forces at play, there are also important differences. Fungicide resistance appears to evolve, in most cases, by de novo point mutations in the target‐site encoding genes; herbicide resistance often evolves through selection of polygenic metabolic resistance from standing variation; and insecticide resistance evolves through a combination of standing variation and de novo mutations in the target site or major metabolic resistance genes. This has practical implications for resistance risk assessment and management, and lessons learnt from pesticide resistance should be applied in the deployment of novel, non‐chemical pest‐control methods.     

Whole-exome sequencing for finding <Emphasis Type="Italic">de novo</Emphasis> mutations in sporadic mental retardation

Recent work has used a family-based approach and whole-exome sequencing to identify de novo mutations in sporadic cases of mental retardation.     

串联质谱图谱从头测序算法研究进展

近年来,基于质谱技术的高通量蛋白质组学研究发展迅速,利用串联质谱图谱鉴定蛋白质是其数据处理中一个基础而又重要的环节．由于不需要利用蛋白质序列数据库,从头测序方法能够分析新物种或者基因组未测序物种的串联质谱数据,具有数据库搜索方法不可替代的优势．简要介绍高通量串联质谱图谱从头测序问题及其研究现状．归纳出几种典型的计算策略并分析了各种策略的优缺点．总结常用的从头测序算法和软件,介绍算法评估的各种指标和常用评估数据集,概括各种算法的特点,展望未来研究可能的发展方向．     

Use of Tetrahymena pyriformis to Evaluate the Effects of Purine and Pyrimidine Analogs1

SYNOPSIS Eighty-four purine and pyrimidine analogs were evaluated for growth inhibition of Tetrahymena pyriformis. The most toxic were 2-fluoroadenine, 2-fluoroadenosine, 6-methylpurine, a series of 5-fluoropyrimidines, and a series of adenine derivatives substituted in the 9-position. 2-Fluoroadenine was metabolized to the ribonucleoside triphosphate and was incorporated into nucleic acids; its inhibition of growth was reversed by high levels of adenine. 6-Methylthiopurine ribonucleoside was phosphorylated, but only to the monophosphate derivative. Contrasting T. pyriformis with mammalian cells gave clues to the mechanism of action of some of the agents. 6-Mercaptopurine, 6-methylthiopurine ribonucleoside, and 6-thioguanine, all potent pseudofeedback inhibitors of de novo purine biosynthesis in mammalian cells, are not toxic to T. pyriformis, which lacks the de novo purine pathway; this implies that inhibition of de novo purine biosynthesis by them underlies their growth inhibition of mammalian cells.     

Changes in genomic methylation patterns during the formation of triploid asexual dandelion lineages

DNA methylation is an epigenetic mechanism that has the potential to affect plant phenotypes and that is responsive to environmental and genomic stresses such as hybridization and polyploidization. We explored de novo methylation variation that arises during the formation of triploid asexual dandelions from diploid sexual mother plants using methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. In dandelions, triploid apomictic asexuals are produced from diploid sexual mothers that are fertilized by polyploid pollen donors. We asked whether the ploidy level change that accompanies the formation of new asexual lineages triggers methylation changes that contribute to heritable epigenetic variation within novel asexual lineages. Comparison of MS‐AFLP and AFLP fragment inheritance in a diploid × triploid cross revealed de novo methylation variation between triploid F₁ individuals. Genetically identical offspring of asexual F₁ plants showed modest levels of methylation variation, comparable to background levels as observed among sibs in a long‐established asexual lineage. Thus, the cross between ploidy levels triggered de novo methylation variation between asexual lineages, whereas it did not seem to contribute directly to variation within new asexual lineages. The observed background level of methylation variation suggests that considerable autonomous methylation variation could build up within asexual lineages under natural conditions.     

Phosphatidylserine synthase and phosphatidylserine decarboxylase are essential for cell wall integrity and virulence in Candida albicans

Phospholipid biosynthetic pathways play crucial roles in the virulence of several pathogens; however, little is known about how phospholipid synthesis affects pathogenesis in fungi such as Candida albicans. A C. albicans phosphatidylserine (PS) synthase mutant, cho1Δ/Δ, lacks PS, has decreased phosphatidylethanolamine (PE), and is avirulent in a mouse model of systemic candidiasis. The cho1Δ/Δ mutant exhibits defects in cell wall integrity, mitochondrial function, filamentous growth, and is auxotrophic for ethanolamine. PS is a precursor for de novo PE biosynthesis. A psd1Δ/Δ psd2Δ/Δ double mutant, which lacks the PS decarboxylase enzymes that convert PS to PE in the de novo pathway, has diminished PE levels like those of the cho1Δ/Δ mutant. The psd1Δ/Δ psd2Δ/Δ mutant exhibits phenotypes similar to those of the cho1Δ/Δ mutant; however, it is slightly more virulent and has less of a cell wall defect. The virulence losses exhibited by the cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ mutants appear to be related to their cell wall defects which are due to loss of de novo PE biosynthesis, but are exacerbated by loss of PS itself. Cho1p is conserved in fungi, but not mammals, so fungal PS synthase is a potential novel antifungal drug target.