Search

Microbial secondary metabolites have long been considered as potential sources of lead compounds for medicinal use due to their rich chemical diversity and extensive biological activities. However, many biosynthetic gene clusters remain silent under traditional laboratory culture conditions, resulting in repeated isolation of a large number of known compounds. The co-culture strategy simulates the complex ecological environment of microbial life by using an ecology-driven method to activate silent gene clusters of microorganisms and tap their metabolic potential to obtain novel bioactive secondary metabolites. In this review, representative studies from 2017 to 2020 on the discovery of novel bioactive natural products from co-cultured microorganisms are summarized. A series of natural products with diverse and novel structures have been discovered successfully by co-culture strategies, including fungus–fungus, fungus–bacterium, and bacterium–bacterium co-culture approaches. These novel compounds exhibited various bioactivities including extensive antimicrobial activities and potential cytotoxic activities, especially when it came to disparate marine-derived species and cross-species of marine strains and terrestrial strains. It could be concluded that co-culture can be an effective strategy to tap the metabolic potential of microorganisms, particularly for marine-derived species, thus providing diverse molecules for the discovery of lead compounds and drug candidates. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42995-020-00077-5) contains supplementary material, which is available to authorized users.

New antibiotics are urgently required in clinical treatment and agriculture with the development of antimicrobial resistance. However, products discovered by repeating previous strategies are either not antibiotics or already known antibiotics. There is a growing demand for efficient strategies to discover new antibiotics. With the continuous improvement of gene sequencing technology and genomic data, some mining strategies have emerged. These strategies are expected to alleviate the current dilemma of antibiotics. In this review, we discuss the recent advances in discovery of bacterial antibiotics from the following aspects: activation of silent gene clusters, genome mining and metagenome mining. In the future, we envision the discovery of natural antibiotic will be accelerated by the combination of these strategies.

A cryptic terpenoid biosynthetic gene cluster ven was characterized from the model actinomycetic strain Streptomyces venezuelae ATCC 15439 through a genome mining approach. Silent gene activation a...

A key role in the communication between fungi and bacteria is played by natural products. Many of their encoding gene clusters are silent under standard laboratory conditions. Interspecies “talk” between microorganisms represents an ecological trigger to activate such silent gene clusters and leads to the formation of novel natural products by the involved species. The understanding of both the activation of silent gene clusters and the ecological function of the produced compounds is of importance to reveal functional microbial interactions required to shape microbiomes.

Natural products (NPs) are critical sources of drug molecules for decades. About two-thirds of natural antibiotics are produced by Streptomyces. Streptomyces have a large number of secondary metabolite biosynthetic gene clusters (SM-BGCs) that may encode NPs. However, most of these BGCs are silent under standard laboratory conditions. Hence, activation of these silent BGCs is essential to current natural products discovery research. In this review, we described the commonly used strategies for silent BGC activation in Streptomyces from two aspects. One focused on the strategies applied in heterologous host, including methods to clone and reconstruct BGCs along with advances in chassis engineering; the other focused on methods applied in native host which includes engineering of promoters, regulatory factors, and ribosomes. With the metabolic network being elucidated more comprehensively and methods optimized more high-thoroughly, the discovery of NPs will be greatly accelerated.

Bioactive secondary metabolites from endophytic fungi provide a robust foundation for novel natural metabolic scaffolds with a plethora of applications. These filamentous fungi contain gene clusters that encode highly significant metabolites; however, many of these clusters remain concealed in laboratory-like settings. Consequently, disparate strategies encompassing genetic manipulation(s) and or metabolomic interventions are obligatory for activating such silent gene clusters and improving metabolite yield. The metabolomic approach basically involves exhaustive exploration and structural information about the cellular metabolome, whereas the genomic approach utilizes complex gene tailoring and refactoring—viz., CRISPR-Cas9—to extract valuable metabolites that are not produced naturally by the indigenous strain. This chapter discusses consolidated utilization of “omics”-based methodologies for sustainable exploitation of bioactive compounds by deploying these ascomycetes.

Constitutively expression of the pathway-specific activators is an effective method to activate silent gene clusters and improve natural product production. In this study, nine shunt products of aminoansamycins (1-9) were identified from a recombinant mutant strain S35-LAL by overexpressed the large-ATP-binding regulator of the LuxR family (LAL) gene aas1 in Streptomyces sp. S35. All the compounds showed no anti-microbial, anti-T3SS and cytotoxic activities.

Fungal secondary metabolites are a wide range of chemical molecules with interesting properties for human applications. Although several metabolites are already used in medicine, agriculture and industry, novel molecules based on unique chemical scaffolds are needed to tackle the challenges of society, such as the increasing antimicrobial resistance.Here, we investigated the secondary metabolism of Penicillium rubens, which is used in industry for the production of β-lactam antibiotics. We elucidated a complex and highly branched pathway responsible for the biosynthesis of the yellow pigment chrysogine, by assigning each cluster gene to biosynthetic steps and metabolic intermediates. Moreover, we explore the potential of this fungus as a platform for the production of heterologous valuable secondary metabolites. The strategies and tools established in this work provide the basis for awakening silent gene clusters and engineering biosynthetic pathways, which could lead to the discovery and development of novel molecules with new biological activities.

New antibiotics are urgently required by human medicine as pathogens emerge with developed resistance to almost all antibiotic classes. Pioneering approaches, methodologies and technologies have facilitated a new era in antimicrobial discovery. Innovative culturing techniques such as iChip and co-culturing methods which use 'helper' strains to produce bioactive molecules have had notable success. Exploiting antibiotic resistance to identify antibacterial producers performed in tandem with diagnostic PCR based identification approaches has identified novel candidates. Employing powerful metagenomic mining and metabolomic tools has identified the antibiotic'ome, highlighting new antibiotics from underexplored environments and silent gene clusters enabling researchers to mine for scaffolds with both a novel mechanism of action and also few clinically established resistance determinants. Modern biotechnological approaches are delivering but will require support from government initiatives together with changes in regulation to pave the way for valuable, efficacious, highly targeted, pathogen specific antimicrobial therapies.

Molecular epigenetic approach activates silent gene cluster producing dimeric bis-spiro-azaphilones in Chaetomium globosum CBS148.51

// Ying Zhou 1, 2, 3 , Nan Song 1, 2 , Xin Li 1, 2, 4 , Ying Han 1, 2, 5 , Zihan Ren 1, 2 , Jing-xian Xu 6 , Yu-chen Han 1, 2 , Fang Li 1, 2, 7 , Xinshan Jia 1, 2 1 Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, 110001, China 2 Department of Pathology, First Affiliated Hospital of China Medical University, Shenyang, 110001, China 3 Department of Emergency, First Affiliated Hospital of China Medical University, Shenyang, 110001, China 4 Department of Physiology, College of Life Science and Biopharmaceutics of Shenyang Pharmaceutical University, Shenyang, 110016, China 5 Department of Pathology, Shenyang Medical College, Shenyang, 110001, China 6 Department of Ophthalmology, The 4th Affiliated Hospital, Eye Institute, China Medical University, The Key Laboratory of Lens Research, Shenyang 110005, China 7 IVF Michigan, Bloomfield Hills, MI, 48304, USA Correspondence to: Xinshan Jia, email: xinshanjia@126.com Keywords: tracheal epithelium, stem cell, Oct3/4, methylation, silent gene Received: February 25, 2016 Accepted: November 21, 2016 Published: December 07, 2016 ABSTRACT We investigated the relationship between promoter methylation and tracheal stem cell activation. We developed a model of rat tracheal epithelium regeneration after 5-fluorouracil (5-FU)-induced injury. Using immunohistochemistry and Western blotting, the expression levels of the stem cell pluripotency regulator Oct3/4 and differentiation marker CK14 were measured after 5-FU treatment. The methylation status of the Oct3/4 , Nanog , and Sox2 promoters was investigated using methylation-specific PCR. Additionally, the effects of 5-azacytidine (5-azaC), a demethylating agent, on Oct3/4 , Nanog , and Sox2 mRNA and protein expression were evaluated. Finally, we measured the activity of the maintenance and de novo DNA methyltransferases DNMT1, DNMT3a, and DNMT3b. Our data indicate that Oct3/4 , Sox2 , and Nanog are transiently expressed in response to 5-FU-induced injury, and then they are gradually silenced as the cells differentiate. DNA methylation can result in silencing of gene expression, and it can determine whether tracheal stem cells are in an active or dormant state. Treatment with 5-FU reversed the methylation of the Oct3/4 , Nanog , and Sox2 promoters, which corresponded to increases in Oct3/4, Nanog, and Sox2 mRNA and protein. Thus, both maintenance and de novo methyltransferases are involved in regulating tracheal stem cell dormancy and activation.

Overexpression of the global regulator LaeA in a marine-derived fungal strain of Penicillium dipodomyis YJ-11 induced obvious morphological changes and metabolic variations. Further chemical investigation of the mutant strain afforded a series of sorbicillinoids including two new ones named 10,11-dihydrobislongiquinolide (1) and 10,11,16,17-tetrahydrobislongiquinolide (2), as well as four known analogues, bislongiquinolide (3), 16,17-dihydrobislongiquinolide (4), sohirnone A (5), and 2&prime, 3&prime, dihydrosorbicillin (6). The results support that the global regulator LaeA is a useful tool in activating silent gene clusters in Penicillium strains to obtain previously undiscovered compounds.

Endophytic fungi live inside vegetal tissues without causing damage to the host plant and may provide lead compounds for drug discovery. The co-culture of two or more endophytic fungi can trigger silent gene clusters, which could lead to the isolation of bioactive compounds. In this study, two endophytic strains isolated from Handroanthus impetiginosus leaves, identified as Talaromyces purpurogenus H4 and Phanerochaete sp. H2, were grown in mixed and axenic cultures. The meroterpenoid austin was detected only in the extracts from the mixed culture. Once isolated, austin displayed very interesting trypanocidal activity, with an IC50 value of 36.6 ± 1.2 μg/mL against Trypanosoma cruzi in the epimastigote form. The results obtained highlight the importance of the co-culturing of endophytic fungi to obtain natural bioactive products. The findings also enhance our understanding of the ecological relationships between endophytic fungi. Fil: do Nascimento, Jainara Santos. Universidade Federal da Bahia; Brasil Fil: Silva, Felipe Moura. Universidade Federal da Bahia; Brasil Fil: Magallanes Noguera, Cynthia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; Argentina Fil: Kurina Sanz, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; Argentina Fil: dos Santos, Elda Gonçalves. Universidade Federal da Bahia; Brasil Fil: Caldas, Ivo Santana. Universidade Federal da Bahia; Brasil Fil: Luiz, Jaine Honorata Hortolan. Universidade Federal da Bahia; Brasil Fil: Silva, Eliane de Oliveira. Universidade Federal da Bahia; Brasil

Covers the period up to 2016 Bacterial-based natural products have long represented a promising resource for the development of commercially relevant therapeutics, and more than two thirds of these products have been developed from members of the genus Streptomyces. The extensive sequencing of bacterial genomes suggests that the majority of gene clusters encoding natural products are silent and not expressed under standard laboratory conditions. However, these clusters can be activated through systematic exchanges between native transcriptionally silent promoters and transcriptionally active promoters. Therefore, the availability of well-studied constitutive and inducible promoters is of the utmost importance for identifying natural products encoded by silent gene clusters. This manuscript provides an overview of the promoter control elements for streptomycetes and examples of their successful application in refactoring the biosynthetic pathways of natural products.

Streptomycetes are the most important source for antibiotics. One representative species is Streptomyces pristinaespiralis–the producer of pristinamycin. Pristinamycin biosynthesis is governed by a complex signalling cascade consisting of seven different transcriptional regulators. Understanding the signalling cascade can help to optimize antibiotic production processes in general but also allows to targetedly activate silent gene cluster expression in order to find new antibiotics.

Here we report an efficient CRISPR-Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in streptomycetes. We applied this one-step strategy to activate multiple BGCs of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromogenes. This potentially scalable strategy complements existing activation approaches and facilitates discovery efforts to uncover new compounds with interesting bioactivities., Graphical abstract

Antibiotic production in Actinobacteria is highly coordinated and subject to diverse environmental and physiological (pre)conditions. Several general factors are known to influence antibiotic production, such as nutrient limitation, growth rate, oxygen supply, or pH conditions. Especially the availability of carbon, phosphate, or nitrogen sources can severely affect antibiotic production. Thereby, the antibiotic production is controlled by pleiotropic, as well as pathway-specific, regulators, both of which we comment on in this chapter. Understanding regulation processes is crucial in order to optimize and manipulate antibiotic production processes. Even more important, it allows to targetedly activate silent gene clusters in order to find new secondary metabolites. The aim of this article is to provide an overview of regulatory factors controlling antibiotic biosynthesis. Furthermore, future perspectives are pointed out on how to exploit them to find new natural compounds.

Microorganisms have provided mankind with a plethora of small molecule natural products ranging from industrial enzymes to therapeutic agents. Analyses of microbial genome sequences have revealed the presence of numerous ‘silent’ or ‘cryptic’ biosynthetic gene clusters (BGCs). Activation of these cryptic biosynthetic pathways can pave way to the discovery of novel bioactive secondary metabolites (SMs). This article summarizes various approaches employed to unlock the hidden biosynthetic potential of microbes and methods developed to study their silent gene products.

Fungi produce a multitude of low-molecular-mass compounds known as secondary metabolites, which have roles in a range of cellular processes such as transcription, development and intercellular communication. In addition, many of these compounds now have important applications, for instance, as antibiotics or immunosuppressants. Genome mining efforts indicate that the capability of fungi to produce secondary metabolites has been substantially underestimated because many of the fungal secondary metabolite biosynthesis gene clusters are silent under standard cultivation conditions. In this Review, I describe our current understanding of the regulatory elements that modulate the transcription of genes involved in secondary metabolism. I also discuss how an improved knowledge of these regulatory elements will ultimately lead to a better understanding of the physiological and ecological functions of these important compounds and will pave the way for a novel avenue to drug discovery through targeted activation of silent gene clusters.

A new case of glucosephosphate isomerase deficiency with mild haemolytic anaemia was observed in a 6-year-old girl. Deficient enzyme was characterized by a profoundly decreased activity in the red cells, a normal electrophoretic phenotype, normal isoelectric point, normal optimum pH, a molecular instability and a clearly decreased Michaelis constant for fructose-6-phosphate. Propositus was double heterozygote for a 'silent gene' inherited from the mother and an abnormal enzyme from the father. Because this abnormal enzyme has undescribed characteristics, it responds to a new variant for which we propose the name GPI-MYTHO.

PHD fingers and bromodomains are found in close proximity to each other in many chromatin-associated proteins and can functionally synergize. Recently, it has been demonstrated that the PHD finger of the KAP1 co-repressor functions as an E3 SUMO ligase for the adjacent bromodomain. This PHD-mediated SUMOylation stabilizes the association of the bromodomain with the chromatin modifiers SETDB1 and the nucleosome remodeling and deacetylation (NuRD) complex, thereby promoting establishment of the silent gene expression state.

Improved antibiotic production and silent gene activation in Streptomyces diastatochromogenes by ribosome engineering

Gene targeting in livestock fibroblasts has proven difficult to achieve, particularly if the target gene is silent. We first tested whether efficient gene targeting at the transcriptionally active ovine alpha1(I) procollagen (COL1A1) locus required the use of a promoter trap vector. We compared gene targeting frequencies at the ovine COL1A1 locus using both a promoter trap and a non-promoter trap selection strategy. We demonstrated that targeted cells could be isolated regardless of whether an enrichment step (promoter trap) was used. Next, we used our optimised protocol to target a non-expressed gene, ovine beta-casein. We obtained clones that were scored positive by PCR for the targeting event, but were negative after cell expansion and Southern analysis. We propose that targeted cells were initially generated but that they were at a selective growth disadvantage during culture. We suggest modifications to the conventional targeting protocol that would prevent such loss of targeted cells.

Most bioactive natural products currently known are synthesized by members of the Actinomycetales order. The development of genetic engineering provides novel genetic tools for the modification of known antibiotics and other bioactive compounds to generate derivatives with improved therapeutic properties. This new technology, named combinatorial biosynthesis, is able of introducing structural modifications in bioactive compounds not easily accessible by chemical means. Furthermore, progress in genome sequencing in this group of microorganisms shows that actinomycetes have a greater potential of synthesizing bioactive compounds than was anticipated. Each genome sequenced shows the presence of 18–37 gene clusters potentially directing the biosynthesis of bioactive compounds that have not been previously identified. Novel strategies are being developed to activate these cryptic or silent gene clusters in these microorganisms, allowing the identification of novel potentially bioactive compounds. This chapter will revise the state of the art in this field of research.

With its expanding toolkit of sophisticated computational and experimental techniques, synthetic biology is currently poised to be a significant contributor to the discovery and development of the next generation of drugs derived from natural products. In this chapter, a number of tools are highlighted which enable the identification, analysis, assembly, and optimization of diverse classes of secondary metabolite gene clusters. Next, the applications of these tools are surveyed, including not only the discovery of new compounds from diverse environments, but also the biosynthesis of new ‘unnatural’ natural products and the activation of otherwise silent gene clusters. Finally, the applications of synthetic biology tools toward the production of valuable drug compounds are illustrated in detail through the examples of erythromycin A, artemisinin, and taxol.

Previously we have shown that the type strain of Lactobacillus acidophilus possesses two S-protein-encoding genes, one of which is silent, on a chromosomal segment of 6 kb. The S-protein-encoding gene in the expression site can be exchanged for the silent S-protein-encoding gene by inversion of this slp segment. In this study the presence of S-protein and corresponding S-protein-encoding genes of strains belonging to species that are closely related to L. acidophilus was determined. All strains investigated were identified by numerical comparison of highly standardized one-dimensional SDS-PAGE whole-cellular-protein patterns. Western blot and Southern blot methods were used to identify the presence of, and homology between, S-proteins and S-protein-encoding genes. From these analyses we conclude that strains of L. acidophilus, L. crispatus, L. amylovorus and L. gallinarum possess an S-layer and contain two slp genes. Strains of L. helveticus possess an S-layer but have only one intact slp gene. Strains of L. gasseri, L. johnsonii and L. delbrueckii subsp. bulgaricus have neither an S-layer nor S-protein-encoding genes hybridizing with probes derived from the L. acidophilus slpA or slpB region. The presence of a highly conserved 5' region in the slp genes of strains of L. acidophilus, L. crispatus, L. amylovorus and L. gallinarum suggests that S-layer variation is a common feature for strains of these species.

Fungi produce numerous secondary metabolites that show antibiotic activity against various microorganisms, antiviral, antitumor and/or fungicidal activity. One of the major challenges, however, is to understand the physiological meaning of the many secondary metabolites for the producing microorganism. As long as this is not understood it is often not possible to produce the compounds under laboratory conditions and to predict the regulatory circuits involved in the regulation of their biosyntheses. This fact is supported by the finding that genome mining of available fungal genomes indicated that their potential to produce compounds is greatly underestimated. Fungal genomes bear the genetic information for the biosynthesis of many more compounds which still await discovery. Despite this limitation, to get access to the vast number of unknown compounds encoded by silent gene clusters, mixing genomic data, genetic engineering and analytical techniques provides a new avenue to discover novel and potentially bioactive natural products.

Seven families segregating apparently silent gene homozygotes have been investigated by enzyme-linked immunosorbent assays. These families confirm not only the heterogeneity of the silent gene in the form of E1s and E1t but in 3 families there is support for the segregation of the new gene E1x.

Silent 型低ChE血症と考えられる一症例の家系検索を行った. 検索の結果, 活性を示すChEは発端者の同胞1名において arylesterase であることが予想されたが他の家族は正常のアロステリック酵素であることが示された. 発端者および同胞1名のアイソザム像はChE-1～ChE-5のすべての band が観察されたがいずれの band も活性の低下がみられ, とくにChE-5の活性低下が著明であった. その他の家族のアイソザイム像は正常であった. 酵素蛋白量の測定では活性発現を示さない酵素蛋白が発端者に認められた. また家族においては予想される酵素蛋白量と測定値は一致した. 以上のことより発端者は type Iと type IIの中間的性質を示すS1 gene, S2 gene の double ヘテロ接合体が推定され, 同胞は type Iのホモ接合体, 第1子および第2子は type Iのヘテロ接合体と推定された.

Activation of fungal silent gene clusters: A new avenue to drug discovery.- Total synthesis studies on macrocyclic pipecolic acid natural products: FK506, the antascomicins and rapamycin.- Application of natural product-inspired diversity-oriented synthesis to drug discovery.- Cheminformatics analysis of natural products: Lessons from nature inspiring the design of new drugs.- Chemical-genetic approaches for exploring the mode of action of natural products.- Epothilones as lead structures for new anticancer drugs - pharmacology, fermentation, and structure-activity-relationships.- Histone deacetylase inhibitors from microorganisms: the Astellas experience.- Ascomycete derivative to MS therapeutic: S1P receptor modulator FTY720.- Artemisinin - an innovative cornerstone for anti-malaria therapy.

Rocket immuno-electrophoresis and enzyme-linked immunosorbent assays were used to estimate the amount of cholinesterase present in 29 apparent silent homozygotes. 26 of these samples were segregated i

In the postgenomic era it has become increasingly apparent that the vast number of predicted biosynthesis genes of microorganisms is not reflected by the metabolic profile observed under standard fermentation conditions. In the absence of a particular (in most cases unknown) trigger these gene loci remain silent. Because these cryptic gene clusters may code for the biosynthesis of important virulence factors, toxins, or even drug candidates, new strategies for their activation are urgently needed to make use of this largely untapped reservoir of potentially bioactive compounds. The discovery of new microbial metabolites through genome mining has proven to be a very promising approach. Even so, the investigation of silent gene clusters is still a substantial challenge, particularly in fungi. Here we report a new strategy for the successful induction of a silent metabolic pathway in the important model organism Aspergillus nidulans, which led to the discovery of novel PKS-NRPS hybrid metabolites.

This study presents additional genetic data on piranha (Serrasalmus rhombeus Linnaeus, 1766) complex previously diagnosed due to the presence of distinct cytotypes 2n = 58 and 2n = 60. Three esterase-D enzyme loci (Est-D1, Est-D2 and Est-D3) were examined and complemented with chromosomal data from 66 piranha specimens collected from Lake Catalão. For all specimens the Est-D1 and Est-D2 loci were monomorphic. In contrast, the Est-D3 locus was polymorphic with genotypes and alleles being differentially distributed in the previously described cytotypes and served as the basis for detecting a new cytotype (2n = 60 B). In cytotype 2n = 58 the Est-D3 locus was also polymorphic and presented Mendelian allelic segregation with four genotypes (Est-D311, Est-D312, Est-D322 and Est-D333) out of six theoretically possible genotypes, presumably encoded by alleles Est-D31 (frequency = 0.237), EsT-D32 (0.710) and Est-D33 (0.053). A Chi-squared (χ2) test for Hardy-Weinberg equilibrium was applied to the Est-D3locus and revealed a genetic unbalance in cytotype 2n = 58, indicating the probable existence in the surveyed area of different stocks for that karyotypic structure. A silent null allele (Est-D30 with a high frequency (0.959) occurred exclusively in the 2n = 60 cytotype. On the other hand, the new cytotype 2n = 60 B described here for the first time was monomorphic for the presumably fixed Est-D33 allele. The data as a whole should contribute to the better understanding the rhombeus complex taxonomic status definitíon in the Central Amazon. © 2006 Sociedade Brasileira de Genética.

Winship Cancer Institute; Emory University School of Medicine; Atlanta, Georgia,USA*Correspondence to: Dipali Sharma; Winship Cancer Institute; Emory UniversitySchool of Medicine; 1365-C; Clifton Road; Room 4060; Atlanta, Georgia 30322USA; Email: dsharma@emory.eduReceived 05/28/04; Accepted 06/24/04Previously published online as a

During thiopentone-fentanyl-nitrous oxide anaesthesia and using a cumulative design, we studied the dose-response relationship of mivacurium in 8 patients: 7 patients phenotypically homozygous for the atypical plasma cholinesterase gene and 1 patient homozygous for the silent gene. The estimated mean ED50 and ED95 were 15 and 20 micrograms.kg.bw-1 in patients homozygous for the atypical gene, and 13 and 16 micrograms.kg.bw-1 in the patient homozygous for the silent gene, respectively. The results indicate that mivacurium is 4-5 times more potent in patients homozygous for the atypical or the silent gene than in patients with normal plasma cholinesterase activity and phenotype.

A silent gene formally designates a rare (non-polymorphic, with frequency below 1%) recessive allele. Therefore, its presence can be unnoticed, even in large samples. The currently used routines in kinship expertise are based on the analysis of autosomal short tandem repeat (STR) loci, which are treated as harbouring just codominant alleles. Only when incompatible results are obtained (like apparent opposite homozygosity), the silent allele is invoked. The main purpose of this work is to quantify the impact of silent alleles in paternity testing in "non-exclusion" situations. We demonstrate that silent alleles can non-negligibly lower the paternity index in trios where opposite homozygosity is observed and we provide some examples in real cases.