TILLING without a plough: a new method with applications for reverse genetics

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetic technique that employs a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms. Its advantages over other reverse genetic techniques include its applicability to virtually any organism, its facility for high-throughput and its independence of genome size, reproductive system or generation time. TILLING is currently being used for the detection of both induced and natural variation in several plant species.     

cid: a rapid and efficient bioinformatic tool for the detection of SSRs from genomic libraries

cid is a computational tool developed in the Web environment to process cloned DNA fragments with the objective of masking the vector and adaptor regions, detecting the presence of microsatellites and designing the most appropriate primer pairs for the amplification of the identified repetitive sequences. This entire process is executed by the user in a simple and automated manner with the data input as a Zip file of chromatograms or a multiFASTA file. Thus, it is possible to analyse dozens of sequences at the same time, optimizing data processing and the search for the information of interest. cid is freely available on http://www.shrimp.ufscar.br/cid/index.php.     

CRISPR: a versatile tool for both forward and reverse genetics research

Human genetics research employs the two opposing approaches of forward and reverse genetics. While forward genetics identifies and links a mutation to an observed disease etiology, reverse genetics induces mutations in model organisms to study their role in disease. In most cases, causality for mutations identified by forward genetics is confirmed by reverse genetics through the development of genetically engineered animal models and an assessment of whether the model can recapitulate the disease. While many technological advances have helped improve these approaches, some gaps still remain. CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated), which has emerged as a revolutionary genetic engineering tool, holds great promise for closing such gaps. By combining the benefits of forward and reverse genetics, it has dramatically expedited human genetics research. We provide a perspective on the power of CRISPR-based forward and reverse genetics tools in human genetics and discuss its applications using some disease examples.     

Highly efficient de novo mutant identification in a Sorghum bicolor TILLING population using the ComSeq approach

Screening large populations for carriers of known or de novo rare single nucleotide polymorphisms (SNPs) is required both in Targeting induced local lesions in genomes (TILLING) experiments in plants and in screening of human populations. We previously suggested an approach that combines the mathematical field of compressed sensing with next‐generation sequencing to allow such large‐scale screening. Based on pooled measurements, this method identifies multiple carriers of heterozygous or homozygous rare alleles while using only a small fraction of resources. Its rigorous mathematical foundations allow scalable and robust detection, and provide error correction and resilience to experimental noise. Here we present a large‐scale experimental demonstration of our computational approach, in which we targeted a TILLING population of 1024 Sorghum bicolor lines to detect carriers of de novo SNPs whose frequency was less than 0.1%, using only 48 pools. Subsequent validation confirmed that all detected lines were indeed carriers of the predicted mutations. This novel approach provides a highly cost‐effective and robust tool for biologists and breeders to allow identification of novel alleles and subsequent functional analysis.     

A practical kinetic model for efficient isolation of useful antibodies from phage display libraries

To isolate phages displaying a practical and useful antibody with a high k_on value and/or a low k_off value from phage display antibody libraries, we developed a rational strategy based on a kinetic model. In the model, the recovery of a phage displaying an antibody after a round of biopanning is expressed as a function of five parameters, the apparent association rate constant of the phage antibody to the immobilized antigen (k_on′), the apparent dissociation rate constant of the phage antibody from the immobilized antigen (k_off′), the effective antigen concentration (C), the time for the binding process (t_b) and the time for the washing process (t_w). An optimum set of operating parameters (C, t_b and t_w) for isolating phages displaying an antibody with a high k_on value was designed based on the model. Three rounds of biopanning were carried out under the designed conditions, against a phage library in which the hypervariable regions of an original antibody were randomized. All isolated phages displayed an antibody with a higher k_on value and one displayed an antibody with a 30-fold greater k_on value than that of the original antibody. Experimental conditions which improve the efficiency of conventional off-rate selections are also described.     

An efficient plating system for rapid isolation of mutants from plant cell suspensions

Summary A plating system for cell suspensions of soybean, SB-1, (Glycine max L. cv. Mandarin) and Datura innoxia D.I. (Mill) was developed using feeder cells. The characteristics of the system are: a) the efficiency of plating (EOP) is high (0.5–0.6), b) over a range of 10–300 plated clumps the EOP is constant, c) the growth rate of plated cells resembles that of suspension cultures (generation time 24 hr.). Clumps with few or with many cells have similar plating efficiencies.Employing the plating system, a mutant resistant to 8 azaguanine (8AG) was isolated from SB-1 in 7 days and purified and tested within an additional 3 weeks. Feeder plates were used to selectively re-isolate 8 AG resistant and maltose utilizing mutants from a 1000-fold excess of wild type cells.The plating technique also can be utilized to isolate auxotrophic mutants since free amino acids are not produced by the feeder suspension. Other applications of this plating technique are discussed.Abbreviations 8AG 8 Azaguanine - 6TG 6 Thioguanine - EMS Ethyl methanesulfonate - EOP Efficiency of plating - HGPRT Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8)     

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: isolation and characterization of a mutant deficient in the activity of phosphoribosylaminoimidazole synthetase.

A new purine-requiring mutant of Chinese hamster ovary cells (CHO-Kl) is described. This mutant, Ade-G, grows on aminoimidazole carboxamide, hypoxanthine, or adenine. It complements all eight of our other previously described Ade- mutants. Biochemical analysis of de novo purine synthesis in whole cells suggests that Ade-G is capable of the first four reactions of de novo purine biosynthesis and that it synthesizes and accumulates phosphoribosylformylglycinamidine (FGAM). Direct enzyme assay in cell-free extracts confirms that Ade-G is defective in phosphoribosylaminoimidazole synthetase activity and does not convert FGAM to phosphoribosylaminoimidazole (AIR), the next intermediate in the de novo biosynthetic pathway.     

Molecular and ultrastructural analysis of a nonchromosomal variegated mutant. Tomato mitochondrial mutants that cause abnormal leaf development.

Mutants were recovered in a population of cybrids formed following protoplast fusion between tomato (Lycopersicon esculentum Mill.) cv UC82 and Lycopersicon pennellii Corr. The cybrids were identified as individuals with recombinant cytoplasmic genomes but only tomato nuclear genomes. The mutants were identified based on two features, a variegated sectoring of light and dark green regions on their leaves, stems, and fruit, and reduced growth in the field. The mutants produced 50% of the shoot fresh weight and 20% of the fruit fresh weight of the parental type, UC82. The variegated sectoring was maternally inherited. The chloroplast genome in the mutants was indistinguishable from the chloroplast genome in UC82, when distribution of restriction endonuclease sites was used as an assay. The mitochondrial genome in the mutants, however, was recombinant, containing genes from UC82 and L. pennellii. Light microscopic analysis of the leaves of the mutants demonstrated an absence of the palisade layer in the variegated sectors. Electron microscopic analysis of these same regions demonstrated an absence of normal inner membranes in the mitochondria of these cells.     

Gamma irradiation as a useful tool for the isolation of astaxanthin-overproducing mutant strains of Phaffia rhodozyma

Astaxanthin is a carotenoid pigment responsible for the red color of the flesh of many marine animals. There is an increasing interest in the use of astaxanthin in aquaculture, chemical, pharmaceutical, and alimentary industries. Phaffia rhodozyma has been identified as the best biological source of astaxanthin. Mutagenesis was carried out using different doses of gamma irradiation (1.0, 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, and 7.0 kGy), and 10 mutant colonies (Gam1-Gam10) were obtained. Highly pigmented mutant strains produced astaxanthin at approximately 15?887.5?μg/L dry mass of yeast, whereas the parental strain produced it at 1061.64?μg/g dry mass of yeast. In the thin-layer chromatography analysis, P. rhodozyma JH-82 and Gam1 mutant strain produced the same retention factor (R(f)) values, but Gam1 showed a higher astaxanthin content than JH-82.     

Progress in TILLING as a tool for functional genomics and improvement of crops

Food security is a global concern and substantial yield increases in crops are required to feed the growing world population. Mutagenesis is an important tool in crop improvement and is free of the regulatory restrictions imposed on genetically modified organisms. Targeting Induced Local Lesions in Genomes(TILLING), which combines traditional chemical mutagenesis with high‐throughput genome‐wide screening for point mutations in desired genes, offers a powerful way to create novel mutant alleles for both functional genomics and improvement of crops. TILLING is generally applicable to genomes whether small or large, diploid or evenallohexaploid, and shows great potential to address the major challenge of linking sequence information to the function of genes and to modulate key traits for plant breeding. TILLING has been successfully applied in many crop species and recent progress in TILLING is summarized below, especially on the developments in mutation detection technology, application of TILLING in gene functional studies and crop breeding. The potential of TILLING/EcoTILLING for functional genetics and crop improvement is also discussed. Furthermore, a small‐scale forward strategy including backcross and selfing was conducted to release the potential mutant phenotypes masked in M2(or M3) plants.     

Establishment of a reverse genetics system for rotavirus

The rotavirus genome is composed of 11 segments of double-stranded RNA (dsRNA). Rotavirus is the leading etiological agent of severe gastroenteritis in infants and young children worldwide. Reverse genetics is the powerful and ideal methodology for the molecular study of virus replication, which enables the virus genome to be artificially manipulated. Very recently, we developed the first reverse genetics system for rotavirus, which enables one to generate an infectious rotavirus containing a novel gene segment derived from cDNA. In this review, we describe each steps of rotavirus replication to understand the background to the establishment of a reverse genetics system for rotavirus, and summarize the reverse genetics systems for segmented dsRNA viruses including rotavirus.     

Progress in TILLING as a tool for functional genomics and improvement of crops

Food security is a global concern and substantial yield increases in crops are required to feed the growing world population. Mutagenesis is an important tool in crop improvement and is free of the regulatory restrictions imposed on genetically modified organisms. Targeting Induced Local Lesions in Genomes（TILLING）, which combines traditional chemical mutagenesis with high‐throughput genome‐wide screening for point mutations in desired genes, offers a powerful way to create novel mutant alleles for both functional genomics and improvement of crops. TILLING is generally applicable to genomes whether small or large, diploid or evenallohexaploid, and shows great potential to address the major challenge of linking sequence information to the function of genes and to modulate key traits for plant breeding. TILLING has been successfully applied in many crop species and recent progress in TILLING is summarized below, especially on the developments in mutation detection technology, application of TILLING in gene functional studies and crop breeding. The potential of TILLING/EcoTILLING for functional genetics and crop improvement is also discussed. Furthermore, a small‐scale forward strategy including backcross and selfing was conducted to release the potential mutant phenotypes masked in M2（or M3） plants.     

A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins

Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.     

Tales from the front lines: the creative essay as a tool for teaching genetics.

In contrast to the more typical mock grant proposals or literature reviews, we describe the use of the creative essay as a novel tool for teaching human genetics at the college level. This method has worked well for both nonmajor and advanced courses for biology majors. The 10- to 15-page essay is written in storylike form and represents a student's response to the choice of 6-8 scenarios describing human beings coping with various genetic dilemmas. We have found this tool to be invaluable both in developing students' ability to express genetic concepts in lay terms and in promoting student awareness of genetic issues outside of the classroom. Examples from student essays are presented to illustrate these points, and guidelines are suggested regarding instructor expectations of student creativity and scientific accuracy. Methods of grading this assignment are also discussed.     

A vector for the removal of deletion mutants from antibody libraries.

To reduce the number of deletion mutants from antibody (Ab) libraries that had been amplified by PCR from peripheral blood lymphocytes, we constructed the Ab expression vector, pLAB, in which DNA coding for a single-chain Ab was inserted into the gene encoding beta-lactamase (Bla) at the 3'-terminus of its signal sequence. After transforming Escherichia coli with this vector, a fusion protein with a functional Bla domain was produced that was able to protect the bacteria from the action of ampicillin (Ap). Libraries can therefore be usefully propagated with this vector, since only those clones carrying inserts that are in frame with Bla will survive Ap selection, while others that carry out-of-frame deletions or internal stop codons are eliminated.     

Cellular genetic study of a somatic instability in a tobacco mutant: in vitro isolation of valine-resistant spontaneous mutants

Summary A chlorophyll-deficient mutant line of tobacco (Nicotiana tabacum), named tl, displays spontaneously on leaves green, white, and twinned green/white somatic variations at high frequencies (10^–3 to 10^–2). The frequency of cell events leading to the somatic variations has been shown to be closely dependent on the stage of differentiation of cells during plant development. The activity of transposable elements is suspected in the tl genotype, and a study of its mutagenic ability was performed by selecting in vitro new mutant cellular types. The cellular marker chosen was the resistance to toxic doses of valine conferred by a permeation deficiency. A reproducible method allowing efficient selection of valine-resistant mutant clones from haploid tobacco mesophyll protoplast-derived cells was used. In 10 out of 12 experiments, the frequency of spontaneous valine-resistant clones obtained with the wild-type control was null for cell populations tested to the 10⁶. On the other hand, spontaneous valine-resistant clones were repeatedly isolated at variable and sometimes high frequencies (greater than 10^–3) from cell populations of the tl type. Valine resistance of plants regenerated from these clones was transmitted to the progeny as a single monogenic mutation. These results indicate an increased mutagenic ability of the tl genotype, as compared to the wild-type line.