Restriction Recognition Sites Research Articles

Having a Same Type IIS Enzyme's Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis.

Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.

Novel mutation in UMPS gene leads to false positive result of DUMPS (genetic disorder) in buffaloes: need for gene sequencing before confirming results of RFLP in new species

Deficiency of uridine monophosphate synthase (DUMPS) is a lethal genetic disorder associated with early embryonic mortality. Murrah and Mehsana male buffaloes (n = 594) were screened for DUMPS by PCR-RFLP technique. A few Murrah buffalo male calves were found to be carriers of DUMPS in RFLP, which has not been reported earlier. On the Sanger sequencing, a novel A to G substitution mutation was identified in AvaI restriction recognition site of UMPS gene in buffaloes. This mutation hinders digestion of DNA by AvaI which leds to false positive results for DUMPS carrier in RFLP. The results indicated that genome sequencing must be performed before confirming results of RFLP in any new species. All the buffaloes that were tested had only wild-type genotype in exon 5 for DUMPS specific allele.

PCR-RFLP Polymorphism of Growth Hormone Gene and Its Association with Growth and Morphometric Traits in Ganjam and Baigani Goats of Odisha

Background: Growth Hormone (GH) gene codes for the growth hormone, an anabolic hormone synthesized and secreted by the somatotroph cells of pituitary. Growth hormone influences many processes such as growth, lactation, reproduction and metabolism. Genetic variation in the gene are utilized as markers for selecting animals that are superior in terms growth, production and reproduction traits. Information available on the polymorphism of growth hormone gene of Ganjam and Baigani goats of Odisha is scanty. The current investigation was done to explore the genetic variation of this gene and its association with morphometric traits. Methods: Genetic polymorphism in exon 2 and 3 of Growth hormone gene in Ganjam goat and Baigani goat was explored with sample size of 100 goats for each. The goats belonged to three locations: Khallikote, Rambha, Chattrapur of Ganjam district. The goats were recorded for their body weights, morphometric traits and morphological traits. Genomic DNA was isolated, the target segment comprising exons 2 and 3 was amplified and PCR-RFLP was carried out using Hae III restriction enzyme. Genotypes were scored. Result: PCR of the locus resulted in 422 bp PCR product. PCR-RFLP using Hae III restriction enzyme yielded only two variants in both the populations. The variant A had only one restriction recognition site on the target gene segment yielding two bands with size of 366bp and 56bp whereas the variant B did not have any restriction site with single band of 422bp. Three genotypes AA, AB and BB were found in both the population. In both the population, the gene and genotype frequency were significantly deviated from the Hardy Weinberg Equilibrium frequency. There was a significant difference in the genotype frequencies of growth hormone gene between Ganjam and Baigani goats. The AB genotype had higher mean value for all morphometric traits than AA and BB genotype though the differences were not found to be significant.

A novel nine base deletion mutation in NADH-cytochrome b5 reductase gene in an Indian family with recessive congenital methemoglobinemia-type-II

Recessive hereditary methemoglobinemia (RCM) associated with severe neurological abnormalities is a very rare disorder caused by NADH- cytochrome b5 reductase (cb5r) deficiency (Type II). We report a case of 11month old male child who had severe mental retardation, microcephaly and gross global developmental delay with methemoglobin level of 61.1%. The diagnosis of NADH-CYB5R3 deficiency was made by the demonstration of significantly reduced NADH-CYB5R3 activity in the patient and intermediate enzyme activity in both the parents. Mutation analysis of the CYB5R gene revealed a novel nine nucleotide deletion in exon 6 leading to the elimination of 3 amino acid residues (Lys173, Ser174 and Val 175). To confirm that this mutation was not an artifact, we performed PCR-RFLP analysis using the restriction enzyme Drd I. As the normal sequence has a restriction recognition site for Drd I which was eliminated by the deletion, a single band of 603-bp was seen in the presence of the homozygous mutation. Molecular modeling analysis showed a significant effect of these 3 amino acids deletion on the protein structure and stability leading to a severe clinical presentation. A novel homozygous 9 nucleotide deletion (p.K173–p.V175del3) is shown to be segregated with the disease in this family. Knowing the profile of mutations would allow us to offer prenatal diagnosis in families with severe neurological disorders associated with RCM — Type II.

PyRAD: assembly of de novo RADseq loci for phylogenetic analyses

Restriction-site-associated genomic markers are a powerful tool for investigating evolutionary questions at the population level, but are limited in their utility at deeper phylogenetic scales where fewer orthologous loci are typically recovered across disparate taxa. While this limitation stems in part from mutations to restriction recognition sites that disrupt data generation, an additional source of data loss comes from the failure to identify homology during bioinformatic analyses. Clustering methods that allow for lower similarity thresholds and the inclusion of indel variation will perform better at assembling RADseq loci at the phylogenetic scale. PyRAD is a pipeline to assemble de novo RADseq loci with the aim of optimizing coverage across phylogenetic datasets. It uses a wrapper around an alignment-clustering algorithm, which allows for indel variation within and between samples, as well as for incomplete overlap among reads (e.g. paired-end). Here I compare PyRAD with the program Stacks in their performance analyzing a simulated RADseq dataset that includes indel variation. Indels disrupt clustering of homologous loci in Stacks but not in PyRAD, such that the latter recovers more shared loci across disparate taxa. I show through reanalysis of an empirical RADseq dataset that indels are a common feature of such data, even at shallow phylogenetic scales. PyRAD uses parallel processing as well as an optional hierarchical clustering method, which allows it to rapidly assemble phylogenetic datasets with hundreds of sampled individuals. Software is written in Python and freely available at http://www.dereneaton.com/software/.

Molecular typing of Coorg black pepper yellows phytoplasma by multiple gene analyses

In previous work, Coorg black pepper yellows phytoplasma (CBPYp), a ‘Candidatus Phytoplasma asteris'-related strain, was identified in association with black pepper plants exhibiting yellows symptoms in southern India. In the present study, multiple gene (16S rRNA, tuf, rplV-rpsC, secY and secA) sequence analyses were carried out for finer characterisation of CBPYp isolates identified in seven plants. Nucleotide sequences of each gene studied were identical among all the CBPYp isolates here analysed. Comparison of virtual restriction fragment length polymorphism (RFLP) patterns, validated by actual digestion of polymerase chain reaction (PCR) products, revealed that CBPYp is a member of subgroups 16SrI-B, rpI-L, tufI-B, secYI-L and secA1-A. Interestingly, alignments of nucleotide sequences with other ‘Candidatus Phytoplasma asteris'-related strains revealed the presence of CBPYp-specific single nucleotide polymorphisms (SNPs), located in restriction sites for endonucleases not used for conventional classification. CBPYp-specific SNPs in genes 16S rRNA, tuf and secA were detectable by virtual and actual RFLP assays, while SNPs present in rplV-rpsC and secY genes were not located in any restriction recognition site. CBPYp-specific SNPs can be used as molecular markers for the specific identification of CBPYp and for future research focused on investigating epidemiology and ecology of CBPYp in India.

An Electron-sharing Network Involved in the Catalytic Mechanism Is Functionally Conserved in Different Glutathione Transferase Classes

In Anopheles dirus glutathione transferase D3-3, there are electrostatic interactions between the negatively charged glutamyl alpha-carboxylate group of glutathione, the positively charged Arg-66, and the negatively charged Asp-100. This ionic interaction is stabilized by a network of hydrogen bonds from Ser-65, Thr-158, Thr-162, and a conserved water-mediated contact. This alternating ionic bridge interaction between negatively and positively charged residues stabilized by a network of hydrogen bonding we have named an electron-sharing network. We show that the electron-sharing network assists the glutamyl alpha-carboxylate of glutathione to function as a catalytic base accepting the proton from the thiol group forming an anionic glutathione, which is a crucial step in the glutathione transferase (GST) catalysis. Kinetic studies demonstrate that the mutation of electron-sharing network residues results in a decreased ability to lower the pKa of the thiol group of glutathione. Although the residues that contribute to the electron-sharing network are not conserved in the primary sequence, structural characterizations indicate that the presence of the network can be mapped to the same region in all GST classes. A structural diversification but functional conservation suggests a significant role for the electron-sharing network in catalysis as the purpose was maintained during the divergent evolution of GSTs. This network appears to be a functionally conserved motif that contributes to the "base-assisted deprotonation" model suggested to be essential for the glutathione ionization step of the catalytic mechanism.

Biosynthesis of an Elastin‐Mimetic Polypeptide with Two Different Chemical Functional Groups within the Repetitive Elastin Fragment

A new protein engineering strategy was utilized to synthesize an elastin-mimetic polypeptide. The primary structure represents an elastic motif composed of thirty amino acids with one lysine and one glutamic acid per repeat unit EMM = (VPGVG VPGKG VGPVG VPGVG VPGEG VPGIG). The gene was constructed using a Seamless Cloning method by generating three DNA cassettes which all encoded the EMM repeat unit, but with different flanking restriction recognition sites. The DNA cassettes were assembled to yield a gene that could be directly cloned into the multiple cloning site of pBluescript II SK+. The resulting gene (EMM)(7) with approximately 650 base pairs in length was further cloned into the expression vector pET-28b. Protein biosynthesis in E. coli strain BLR(DE3) resulted in the 21.5 kDa repeating polypeptide His(6)-(EMM)(7) yielding up to 50 mg . L(-1) of cell culture. Secondary structure analysis by far UV circular dichroism revealed a minimum at 197 nm and a shoulder at 218 nm indicative for a random coil with some type II beta-turn conformation content. Lower critical solution temperature (LCST) behavior strongly depends on salt and polypeptide concentration. Importantly, first cross-linking experiments indicate successful hydrogel formation with a surface structure reminiscent to natural elastin as visualized by SEM micrographs.

Prenatal molecular evaluation of six fetuses in four unrelated Korean families with ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) deficiency, an X-linked inborn error of the urea cycle, leads to the accumulation of ammonia, causing neurologic deficits. Clinical management for the patients with OTC deficiency is frustrating and requires a burdensome medical regimen, since they may have impairment and recurrent episodes of hyperammonemia in spite of intensive care. Therefore, prenatal diagnosis of the affected fetus is important in genetic counselling for the family at high risk. In this study, mutations in the OTC gene of three obligate heterozygotes and a proband have been identified in four unrelated families: R141Q, R320X, H214Y, M205T. Each mutation altered restriction recognition sites; TaqI for R141Q, NlaIII for M205T, RsaI for H214Y, BclI for R320X. Based on their molecular defects, prenatal diagnoses of 6 fetuses including one set of fraternal twins were successfully made at the ninth to eleventh week of gestation by polymerase chain reaction (PCR)-restriction digestion using genomic DNA from chorionic villus sampling (CVS). We predicted the outcome of all fetuses prenatally. Among six, four were females and two were males, which were determined by PCR amplification of the sex determining region of the Y chromosome (SRY) gene. Each carried a wild type allele for the corresponding mutant allele. They were also tested postnatally for the mutations to be unaffected.

Genetic diagnosis of familial hypercholesterolemia in affected relatives using pedigree tracing

Objectives: To describe a process of diagnosing familial hypercholesterolemia (FH) at the DNA level in selected family members of affected individuals. Design and Methods: A 63-year-old male patient presented with cholesterol elevations consistent with heterozygous familial hypercholesterolemia. Through participation with the international “MED-PED FH” project to detect affected relatives and to identify their LDL-receptor mutation, the patient was discovered to carry the Lebanese mutation, whereby the codon for cysteine at residue 660 instead codes for a premature termination (C660X), thus truncating the protein product. This mutation also created a new restriction recognition site for the endonuclease Hinfl, which permitted rapid detection of the mutation in selected family members using restriction fragment-length polymorphisms. Results: The patient's son, who had cholesterol levels consistent with heterozygous FH, was also found to be a heterozygote for the C660X variant of the LDL-receptor. Conclusions: Diagnosis of familial hypercholesterolemia at the DNA level is possible as a relatively rapid screening technique in families with a known LDL-receptor mutation, established through participation with the MED-PED FH project.

Recombinant leech-derived tryptase inhibitor: construction, production, protein chemical characterization and inhibition of HIV-1 replication.

A synthetic gene coding for leech-derived tryptase inhibitor, form C (LDTI-C), was designed, cloned and expressed. The gene assembled via 6 oligonucleotides contains linker sequences, stop codons and internal restriction recognition sites for cloning, expression and cassette mutagenesis. Periplasmatic expression products could not be detected in Escherichia coli (E. coli), but strong expression was found using Saccharomyces cerevisiae (S. cerevisiae) ( > 10 mg/l culture broth) if a variant of pVT102U/alpha was used as vector. The secreted material was isolated after cross-flow filtration and purified by cation exchange chromatography. The recombinant material proved to be pure and homogeneous by electrophoretic and chromatographic analyses. Amino acid sequencing and molecular mass determination (4737.6 +/- 0.77 Da) by electrospray ionization mass spectrometry confirmed that rLDTI-C was processed correctly and that it is indistinguishable from LDTI-C. The far UV-CD (circular dichroism) spectrum of the recombinant inhibitor is typical for a small folded protein. rLDTI-C is inhibitorily fully active, its complexes with bovine trypsin and human mast cell tryptase display equilibrium dissociation constants which are nearly identical to those with the natural inhibitor. Remarkably, the inhibitor blocked replication of HIV-1 in HUT-78 cells at a concentration of 20 microM.

Genotypic analysis of mutations in Taq I restriction recognition sites by restriction fragment length polymorphism/polymerase chain reaction.

Point mutations in somatic cells play a role in the etiology of several classes of human pathologies. Experimental procedures are required that allow the detection and quantitation of such mutations in disease-related genes in tissue biopsy samples without the need for the selection of mutated cells. We describe the genotypic analysis of single base pair mutations in the Taq I endonuclease recognition sequence TCGA, residues 2508-2511 of exon 2 of the human c-H-ras1 gene, by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) approach. The high thermostability of Taq I endonuclease allows the continuous removal of eventual residual wild-type sequences during the thermocycling of the PCR and reduces polymerase errors in the final RFLP/PCR product to a minimum. As few as five copies of a mutant standard containing two base pair changes in the chosen Taq I site could be rescued from 10(8) copies of wild-type DNA. Taq I RFLP/PCR holds promise for the monitoring of mutations in biochemical epidemiology.

Diagnostic approaches to renal genetic disorders using DNA analysis.

Recent developments in molecular genetic technology have made it possible to diagnose many genetic disorders affecting the kidney before they are clinically manifest. For a disorder to be diagnosed by DNA analysis, either the causative gene must be known and cloned, or a closely linked DNA segment must have been identified. If one of these criteria is met, the disorder may be diagnosed either by direct detection of a mutation, if it is known, or indirectly by linkage analysis of the region using closely linked genetic markers. The methodology currently employed for direct detection of mutation includes the Southern blot, which will detect large structural alterations of genes or mutations altering a restriction recognition site, or the use of allele-specific oligonucleotides, which will detect specific point mutations. Linkage analysis is performed on DNA from multiple family members of the person at risk. Polymorphic markers are "tracked" in the family to determine the allele segregating with the disease gene. These methods are now routinely applied to the diagnosis of mendelian disorders affecting the kidney. It is anticipated that progress over the next decade will extend these applications to detection of the genetic component(s) contributing to multifactorial conditions.

Potassium permanganate as an in situ probe for B-Z and Z-Z junctions.

The availability of DNA structural probes that can be applied to living cells is essential for the analysis of biological functions of unusual DNA structures adopted in vivo. We have developed a chemical probe assay to detect and quantitate left-handed Z-DNA structures in recombinant plasmids in growing E. coli cells. Potassium permanganate selectively reacts with B-Z or Z-Z junction regions in supercoiled plasmids harbored in the cells. Restriction enzyme recognition sites located at these junctions are not cleaved by the corresponding endonuclease after modification with KMnO4. This inhibition of cleavage allows the determination of the relative amounts of B- and Z-forms of the cloned inserts inside the cell. We have successfully applied this method to monitor the extent of Z-DNA formation in E. coli as a function of the growth phase and mutated topoisomerase or gyrase activities. The assay can in principle be used for any unusual DNA structure that contains a restriction recognition site inside or near the structural alteration. It can be a useful tool to analyze in vivo correlations between DNA structure and gene regulatory events.

Prenatal diagnosis of inherited hemoglobinopathies.

This paper reviews the methodology available to make prenatal diagnosis of inherited hemoglobinopathies by DNA analysis and the strategy to be used for the large scale application of this procedure to high-risk populations. The most straightforward approach for prenatal diagnosis is nowadays based on the analysis of DNA enzymatically amplified by the polymerase chain reaction (PCR). The mutations, produced by gross structural rearrangement of the DNA and those affecting a restriction recognition site, are directly detected by visualization following ethidium bromide staining of the electrophoretic pattern resulting from enzymatic digestion of amplified DNA. The remaining ones are detected by dot blot analysis with allelic specific oligonucleotide probes. Because in each population a limited number of specific beta-thalassemia mutations are prevalent, prenatal diagnosis by DNA analysis may be carried out by a population-specific strategy based on the amplification of those regions of the beta-globin genes containing the mutations most frequently occurring in each population followed by dot blot analysis with allelic specific oligonucleotide probes. This approach has the great advantage of being very simple, because radioactive probes are not necessary, very rapid, the results being obtained within 24 hours from sampling and very sensitive, only a limited amount of DNA in the order of 50 ng being necessary.

Repair of thymine.guanine and uracil.guanine mismatched base-pairs in bacteriophage M13mp18 DNA heteroduplexes.

Repair of thymine.guanine (T.G) and uracil.guanine (U.G) mismatched base-pairs in bacteriophage M13mp18 replicative form (RF) DNA was compared upon transfection into repair-proficient or repair-deficient Escherichia coli strains. Oligonucleotide-directed mutagenesis was used to prepare covalently closed circular heteroduplexes that contained the mismatched base-pair at a restriction recognition site. The heteroduplexes were unmethylated at dam (5'-GATC-3') sites to avoid methylation-directed biasing of repair. In an E. coli host containing uracil-DNA glycosylase (ung+), about 97% of the transfecting U.G-containing heteroduplexes had the U residue excised by the uracil-excision repair system. With the analogous T.G mispair, mismatch repair operated on almost all of the transfecting heteroduplexes and removed the T residue in about 75% of them when the mismatched T was on the minus strand of the RF DNA. Similar preferential excision of the minus-strand's mismatched base was observed whether the heteroduplex RF DNA molecules had only one or both strands unmethylated at dcm (5'-CC(A/T)GG-3') sites and whether the RF DNA was prepared by primer extension in vitro or by reannealing mutant and non-mutant DNA strands. Also, the extent and directionality of repair was the same at a U.G mispair in ung- host cells as at the analogous T.G mispair in ung- or ung+ cells. Only in a mismatch repair-deficient (mutH-) host was the plus strand of the transfecting M13mp18 heteroduplex DNA preferentially repaired. It is suggested that the plus strand nick made by the M13-encoded gene II protein might be employed by a mutH- host to initiate repair on that strand.

Restriction of λ trp bacteriophages by Escherichia coli K

trp-transducing derivatives of phage λ have been used to study Escherichia coli K specific restriction in vivo. The expression of the trp genes from unmodified phages during infection of a rec +, restricting host is eliminated by restriction. In a K-restricting recB,C host, where degradation of restricted phage DNA is prevented, expression of the trp genes is little affected by the presence of a single unmodified, K-restriction recognition site, even when that site is within the trpE gene. RI restriction, in contrast to K restriction, prevents trp gene expression in a recB,C host when the restriction target is between the trp genes and the relevant promoter. The presence of two K-restriction recognition sites in a λ trp phage can have a marked effect on trp gene expression. This effect can be interpreted as the result of preferential breakage between the two restriction recognition sites. We conclude that K restriction does not break susceptible DNA at, or even preferentially near, a restriction recognition sequence.

The trpE gene of Escherichia coli K contains a recognition sequence for the K-restriction system

A recognition sequence for the K-restriction system has been localized within the trpE gene of Escherichia coli. Mutations conferring resistance to restriction do not always inactivate the E gene. By selecting for phage mutants which have simultaneously lost two restriction-recognition sites, one in trpE and the other near gene N of phage lambda, we have isolated deletions which localize a site of action of the N protein between cIII and N itself.