Jorge Yáñez

Combination of DMT-mononucleotide and Fmoc-trinucleotide phosphoramidites in oligonucleotide synthesis affords an automatable codon-level mutagenesis method

BACKGROUND Synthetic DNA has been used to introduce variability into protein-coding regions. In protocols that produce a few mutations per gene, the sampling of amino-acid sequence space is limited by the bias imposed by the genetic code. It has long been apparent that the incorporation of trinucleotides in the synthetic regime would circumvent this problem and significantly enhance the usefulness of the technique. RESULTS A new method is described for the creation of codon-level degenerate oligodeoxyribonucleotides that combines conventional dimethoxytrityl (DMT) mononucleoside phosphoramidite chemistry with 9-fluorenylmethoxycarbonyl (Fmoc) trinucleotide phosphoramidites (whose synthesis is reported in the paper). The substoichiometric use of these Fmoc-trinucleotides in an automatable, solid-phase synthesis procedure afforded DNA fragments comprising the wild-type sequence and a controllable distribution of mutants within two- and three-codon stretches of DNA, within the multiple cloning site of the conventional cloning vector pUC19. CONCLUSIONS DMT and Fmoc are compatible protecting groups in conventional oligonucleotide synthesis methods, resulting in controllable levels of codon-based mutagenesis.

TrimerDimer: an oligonucleotide-based saturation mutagenesis approach that removes redundant and stop codons

9-fluorenylmethoxycarbonyl (Fmoc) and 4,4′-dimethoxytrityl (DMTr) are orthogonal hydroxyl protecting groups that have been used in conjunction to assemble oligonucleotide libraries whose variants contain wild-type and mutant codons randomly interspersed throughout a focused DNA region. Fmoc is labile to organic bases and stable to weak acids, whereas DMTr behaves oppositely. Based on these chemical characteristics, we have now devised TrimerDimer, a novel codon-based saturation mutagenesis approach that removes redundant and stop codons during the assembly of degenerate oligonucleotides. In this approach, five DMTr-protected trinucleotide phosphoramidites (dTGG, dATG, dTTT, dTAT and dTGC) and five Fmoc-protected dinucleotide phosphoramidites (dAA, dTT, dAT, dGC and dCG) react simultaneously with a starting oligonucleotide growing on a solid support. The Fmoc group is then removed and the incorporated dimers react with a mixture of three DMTr-protected monomer phosphoramidites (dC, dA and dG) to produce 15 trinucleotides: dCAA, dAAA, dGAA, dCTT, dATT, dGTT, dCAT, dAAT, dGAT, dCGC, dAGC, dGGC, dCCG, dACG and dGCG. After one mutagenic cycle, 20 codons are generated encoding the 20 natural amino acids. TrimerDimer was tested by randomizing the four contiguous codons that encode amino acids L64–G67 of an engineered, nonfluorescent GFP protein. Sequencing of 89 nonfluorescent mutant clones and isolation of two fluorescent mutants confirmed the principle.

Molecular epidemiology and genetic diversity of Entamoeba species in a chelonian collection

Veterinary medicine has focused recently on reptiles, due to the existence of captive collections in zoos and an increase in the acquisition of reptiles as pets. The protozoan parasite, Entamoeba can cause amoebiasis in various animal species and humans. Although amoebiasis disease is remarkably rare in most species of chelonians and crocodiles, these species may serve as Entamoeba species carriers that transmit parasites to susceptible reptile species, such as snakes and lizards, which can become sick and die. In this study, we identified the Entamoeba species in a population of healthy (disease-free) chelonians, and evaluated their diversity through the amplification and sequencing of a small subunit rDNA region. Using this procedure, three Entamoeba species were identified: Entamoeba invadens in 4.76 % of chelonians, Entamoeba moshkovskii in 3.96 % and Entamoeba terrapinae in 50 %. We did not detect mixed Entamoeba infections. Comparative analysis of the amplified region allowed us to determine the intra-species variations. The E. invadens and E. moshkovskii strains isolated in this study did not exhibit marked differences with respect to the sequences reported in GenBank. The analysis of the E. terrapinae isolates revealed three different subgroups (A, B and C). Although subgroups A and C were very similar, subgroup B showed a relatively marked difference with respect to subgroups A and C (Fst = 0.984 and Fst = 1.000, respectively; 10-14 % nucleotide variation, as determined by blast) and with respect to the sequences reported in GenBank. These results suggested that E. terrapinae subgroup B may be either in a process of speciation or belong to a different lineage. However, additional research is necessary to support this statement conclusively.

A new subtype of Entamoeba gingivalis: “E. gingivalis ST2, kamaktli variant”

Entamoeba gingivalis is a protozoan that resides in the oral cavity. Using molecular biology techniques, we identified a novel organism that shares the same ecological niche as E. gingivalis. To differentiate this organism from E. gingivalis, we named it “kamaktli variant.” By sequencing the 18S-ITS1-5.8S-ITS2 rRNA region, we demonstrated that kamaktli variant is 89% identical to E. gingivalis. To elucidate the relationship between kamaktli variant and E. gingivalis, we performed a phylogenetic analysis. Both taxa clustered in the same clade with high support, indicating that the amoebas are closely related (98/99/1.00, maximum parsimony/maximum likelihood/MrBayes, respectively). Given this information, we propose that these molecular differences between kamaktli variant and E. gingivalis ST1 are sufficient to distinguish them as independent subtypes, and we name the new subtype “E. gingivalis ST2, kamaktli variant.”

Prevalence of two Entamoeba gingivalis ST1 and ST2-kamaktli subtypes in the human oral cavity under various conditions

Advances in molecular biology have facilitated analyses of the oral microbiome; however, the parasites role is poorly understood. Periodontal disease is a multifactorial process involving complex interactions among microorganisms, the host, and environmental factors. At present, the precise composition of the mouth parasites microbiota is unclear. Two protozoan species have been detected in the oral microbiota: Trichomonas tenax and Entamoeba gingivalis, and a new variant, E. gingivalis-ST2-kamaktli, was recently identified by us. In this study, both E. gingivalis and the new E. gingivalis-ST2-kamaktli variant were detected in the oral cavities of people with healthy periodontium, individuals undergoing orthodontic treatment, and patients with periodontal disease. In the group with healthy periodontium, the prevalence of E. gingivalis-ST1 was 48.6% and that of E. gingivalis-ST2-kamaktli 29.5%, with a combined prevalence of 54.3%. In patients undergoing orthodontics treatment, 81.2% carried both amoebas, with 47.5% having E. gingivalis-ST1 and 73.8% E. gingivalis-ST2-kamaktli. In people with periodontal disease, the prevalence of E. gingivalis-ST1 was 57.8%, and that of E. gingivalis-ST2-kamaktli 50.0%, with a combined prevalence of 73.5%; hence, E. gingivalis-ST1 and E gingivalis-ST2-kamaktli were detected in all three groups. The question arises, what are E. gingivalis-ST1 and E. gingivalis-ST2-kamaktli doing in the oral cavity? Although, the answer remains unclear, our results suggest that each amoeba subtype is genetically distinct, and they exhibit different patterns of infectious behavior. We hypothesize that E. gingivalis-ST1 and E. gingivalis-ST2-kamaktli may represent separate species. Our data contribute to better understanding of the roles of E. gingivalis-ST1 and E. gingivalis-ST2-kamaktli in the oral microbiota.

CiPerGenesis, a Mutagenesis Approach that Produces Small Libraries of Circularly Permuted Proteins Randomly Opened at a Focused Region: Testing on the Green Fluorescent Protein.

Circularly permuted proteins (cpPs) represent a novel type of mutant proteins with original termini that are covalently linked through a peptide connector and opened at any other place of the polypeptide backbone to create new ends. cpPs are finding wide applications in biotechnology because their properties may be quite different from those of the parental protein. However, the actual challenge for the creation of successful cpPs is to identify those peptide bonds that can be broken to create new termini and ensure functional and well-folded cpPs. Herein, we describe CiPerGenesis, a combinatorial mutagenesis approach that uses two oligonucleotide libraries to amplify a circularized gene by PCR, starting and ending from a focused target region. This approach creates small libraries of circularly permuted genes that are easily cloned in the correct direction and frame using two different restriction sites encoded in the oligonucleotides. Once expressed, the protein libraries exhibit a unique sequence diversity, comprising cpPs that exhibit ordinary breakpoints between adjacent amino acids localized at the target region as well as cpPs with new termini containing user-defined truncations and repeats of some amino acids. CiPerGenesis was tested at the lid region G134-H148 of green fluorescent protein (GFP), revealing that the most fluorescent variants were those starting at Leu141 and ending at amino acids Tyr145, Tyr143, Glu142, Leu141, Lys140, and H139. Purification and biochemical characterization of some variants suggested a differential expression, solubility and maturation extent of the mutant proteins as the likely cause for the variability in fluorescence intensity observed in colonies.

Two novel mutations in ZAP70 gene that result in human immunodeficiency

• Two novel mutations in ZAP70 in a patient with an early-onset immunodeficiency are described.

Spiked Genes: A Method to Introduce Random Point Nucleotide Mutations Evenly throughout an Entire Gene Using a Complete Set of Spiked Oligonucleotides for the Assembly

In vitro mutagenesis methods have revolutionized biological research and the biotechnology industry. In this study, we describe a mutagenesis method based on synthesizing a gene using a complete set of forward and reverse spiked oligonucleotides that have been modified to introduce a low ratio of mutant nucleotides at each position. This novel mutagenesis scheme named “Spiked Genes” yields a library of clones with an enhanced mutation distribution due to its unbiased nucleotide incorporation. Using the far-red fluorescent protein emKate as a model, we demonstrated that Spiked Genes yields richer libraries than those obtained via enzymatic methods. We obtained a library without bias toward any nucleotide or base pair and with even mutations, transitions, and transversion frequencies. Compared with enzymatic methods, the proposed synthetic approach for the creation of gene libraries represents an improved strategy for screening protein variants and does not require a starting template.