Genomic sequencing by ligation-mediated PCR

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited due to the complexity of the mammalian genome. Ligation-mediated PCR (LMPCR) is a sensitive genomic sequencing procedure that generates high quality, reproducible sequence ladders starting with only 1 μg of uncloned mammalian DNA per reaction. This genomic sequencing procedure can be adapted for various methylation, in vivo footprinting and DNA adduct mapping procedures. We provide a detailed protocol for genomic sequencing by LMPCR and discuss the principles and applications of the method.     

Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.     

Phosphophoryn,a biomineralization template protein: pH-dependent protein folding experiments

The protein folding behavior of a polyelectrolyte protein, bovine dentine phosphophoryn (BDPP), in the pH range of 1.82–11.0 has been investigated. One- and two-dimensional nmr spectroscopy has been utilized to obtain proton spin assignments for amino acid residues in D₂O and in H₂O. One-dimensional ³¹P-nmr experiments verify the existence of three separate classes of O-phosphoserine (PSer) resonances in BDPP (α, β, χ), representing three distinct PSer residue populations at pH 6.94. By means of pH titration and ¹H-nmr, five populations of Asp residues can be identified. Three of these populations exhibit secondary inflection points on their pH titration curves that correspond to an observed pK_a of 6.17–6.95. The presence or absence of secondary inflection points for Asp populations and the ³¹P-nmr chemical shift dispersion for the three PSer residue populations indicate that BDPP may be comprised of homologous (Asp-Asp)_n. (PSer-PSer)_n, and heterologous (PSer-Asp)_n sequences arranged into polyelectrolyte cluster regions. The pH titration also revealed that certain populations of Ser, Gly, and Pro residues in BDPP exhibit pH-dependent resonance frequency shifts. The “apparent” pK, for the transition points of these frequency shifts corresponds to either the pK of Pser monophosphatc ester and/or the pK_a of Asp COOH group of BDPP polyelectrolyte regions. On the basis of these transition points, we can assign four types of Ser, Gly, or Pro-containing “intervening” regions in BDPP, based on their sensitivity to protonation and deprotonation events occurring at (Asp)_n, (PSer)_n, or (PSer-Asp)_n anionic cluster regions that flank the intervening regions. Our ¹H-ninr experiments also reveal that BDPP assumes a folded conformation at low pH. As the pH increases, this conformation undergoes several unfolding transitions as the BDPP molecule assumes more open conformations in response to increased electrostatic repulsion between polyelectrolyte anionic regions in the protein. These folding-unfolding transitions are mediated by the intervening regions, which act as “hinges” to allow the polyelectrolyte regions to fold relative to one another. © 1994 John Wiley & Sons, Inc.     

A program for template matching of protein sequences

The matching of a template to a protein sequence is simplifiedby treating it as a special case of sequence alignment. Restrictionof the distances between motifs in the template controls againstspurious matches within very long sequences. The program usingthis algorithm is fast enough to be used in scanning large databasesfor sequences matching a complex template. Received on August 17, 1987; accepted on January 11, 1988     

Analysis of microRNA signatures using size-coded ligation-mediated PCR

The expression pattern and regulatory functions of microRNAs (miRNAs) are intensively investigated in various tissues, cell types and disorders. Differential miRNA expression signatures have been revealed in healthy and unhealthy tissues using high-throughput profiling methods. For further analyses of miRNA signatures in biological samples, we describe here a simple and efficient method to detect multiple miRNAs simultaneously in total RNA. The size-coded ligation-mediated polymerase chain reaction (SL-PCR) method is based on size-coded DNA probe hybridization in solution, followed-by ligation, PCR amplification and gel fractionation. The new method shows quantitative and specific detection of miRNAs. We profiled miRNAs of the let-7 family in a number of organisms, tissues and cell types and the results correspond with their incidence in the genome and reported expression levels. Finally, SL-PCR detected let-7 expression changes in human embryonic stem cells as they differentiate to neuron and also in young and aged mice brain and bone marrow. We conclude that the method can efficiently reveal miRNA signatures in a range of biological samples.     

Identification of protein sequence homology by consensus template alignment

A pattern-matching procedure is described, based on fitting templates to the sequence, which allows general structural constraints to be imposed on the patterns identified. The templates correspond to structurally conserved regions of the sequence and were initially derived from a small number of related sequences whose tertiary structures are known. The templates were then made more representative by aligning other sequences of unknown structure. Two alignments were built up containing 100 immunoglobulin variable domain sequences and 85 constant domain sequences, respectively. From each of these extended alignments, templates were generated to represent features conserved in all the sequences. These consisted mainly of patterns of hydrophobicity associated with beta-structure. For structurally conserved beta-strands with no conserved features, templates based on general secondary structure prediction principles were used to identify their possible locations. The specificity of the templates was demonstrated by their ability to identify the conserved features in known immunoglobulin and immunoglobulin-related sequences but not in other non-immunoglobulin sequences.     

Gene ontology improves template selection in comparative protein docking

Structural characterization of protein-protein interactions is essential for our ability to study life processes at the molecular level. Computational modeling of protein complexes (protein docking) is important as the source of their structure and as a way to understand the principles of protein interaction. Rapidly evolving comparative docking approaches utilize target/template similarity metrics, which are often based on the protein structure. Although the structural similarity, generally, yields good performance, other characteristics of the interacting proteins (eg, function, biological process, and localization) may improve the prediction quality, especially in the case of weak target/template structural similarity. For the ranking of a pool of models for each target, we tested scoring functions that quantify similarity of Gene Ontology (GO) terms assigned to target and template proteins in three ontology domains—biological process, molecular function, and cellular component (GO-score). The scoring functions were tested in docking of bound, unbound, and modeled proteins. The results indicate that the combined structural and GO-terms functions improve the scoring, especially in the twilight zone of structural similarity, typical for protein models of limited accuracy.     

Expressed protein ligation to study protein interactions: semi-synthesis of the G-protein alpha subunit

Interactions between G proteins and GPCRs are fundamental for transmitting signals for a multitude of physiologic responses. Little is known regarding the protein-protein interface between the G protein and the receptor, much less the mechanisms for receptor activation of G proteins. Here, we will describe how expressed protein ligation will aid in the study of protein-protein interactions between semi-synthetic G alpha subunits and GPCRs.     

Protein nucleation and crystallization by homologous protein thin film template

A new method of protein nucleation and crystallization based on Langmuir-Blodgett technology is here utilized for the template stimulation of crystal growth of so far non-crystallized proteins. Microcrystals (60-120 microm) of bovine cytochrome P450scc and human protein kinase CKII alpha subunit were obtained with use of the homologous protein thin film template by vapor diffusion modified hanging drop method. The induction of microcrystals nucleation by the thin template confirms in the two different important classes of proteins, until now never crystallized, the positive stimulatory influence for crystal formation of protein thin film template, which was observed in an earlier study with a model system (chicken egg white lysozyme) as an unexpected acceleration and enhancement in the crystal growth.     

Scoring function for automated assessment of protein structure template quality

We have developed a new scoring function, the template modeling score (TM-score), to assess the quality of protein structure templates and predicted full-length models by extending the approaches used in Global Distance Test (GDT)1 and MaxSub.2 First, a protein size-dependent scale is exploited to eliminate the inherent protein size dependence of the previous scores and appropriately account for random protein structure pairs. Second, rather than setting specific distance cutoffs and calculating only the fractions with errors below the cutoff, all residue pairs in alignment/modeling are evaluated in the proposed score. For comparison of various scoring functions, we have constructed a large-scale benchmark set of structure templates for 1489 small to medium size proteins using the threading program PROSPECTOR_3 and built the full-length models using MODELLER and TASSER. The TM-score of the initial threading alignments, compared to the GDT and MaxSub scoring functions, shows a much stronger correlation to the quality of the final full-length models. The TM-score is further exploited as an assessment of all 'new fold' targets in the recent CASP5 experiment and shows a close coincidence with the results of human-expert visual assessment. These data suggest that the TM-score is a useful complement to the fully automated assessment of protein structure predictions. The executable program of TM-score is freely downloadable at http://bioinformatics.buffalo.edu/TM-score.     

Improving protein template recognition by using small-angle x-ray scattering profiles

Small-angle x-ray scattering (SAXS) is able to extract low-resolution protein shape information without requiring a specific crystal formation. However, it has found little use in atomic-level protein structure determination due to the uncertainty of residue-level structural assignment. We developed a new algorithm, SAXSTER, to couple the raw SAXS data with protein-fold-recognition algorithms and thus improve template-based protein-structure predictions. We designed nine different matching scoring functions of template and experimental SAXS profiles. The logarithm of the integrated correlation score showed the best template recognition ability and had the highest correlation with the true template modeling (TM)-score of the target structures. We tested the method in large-scale protein-fold-recognition experiments and achieved significant improvements in prioritizing the best template structures. When SAXSTER was applied to the proteins of asymmetric SAXS profile distributions, the average TM-score of the top-ranking templates increased by 18% after homologous templates were excluded, which corresponds to a p-value < 10⁻⁹ in Student's t-test. These data demonstrate a promising use of SAXS data to facilitate computational protein structure modeling, which is expected to work most efficiently for proteins of irregular global shape and/or multiple-domain protein complexes.     

Multiplex single nucleotide polymorphism genotyping by adapter ligation-mediated allele-specific amplification

An improved approach for increasing the multiplex level of single nucleotide polymorphism (SNP) typing by adapter ligation-mediated allele-specific amplification (ALM-ASA) has been developed. Based on an adapter ligation, each reaction requires n allele-specific primers plus an adapter-specific primer that is common for all SNPs. Thus, only n+1 primers are used for an n-plex PCR amplification. The specificity of ALM-ASA was increased by a special design of the adapter structure and PCR suppression. Given that the genetic polymorphisms in the liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) have profound effects on responses of individuals to a particular drug, we selected 17 SNPs in the CYP2D6 gene as an example for the multiplex SNP typing. Without extensive optimization, we successfully typed 17-plex SNPs in the CYP2D6 gene by ALM-ASA. The results for genotyping 70 different genome samples by the 17-plex ALM-ASA were completely consistent with those obtained by both Sanger's sequencing and PCR restriction fragment length polymorphism (PCR-RFLP) analysis. ALM-ASA is a potential method for SNP typing at an ultra-low cost because of a high multiplex level and a simple optimization step for PCR. High-throughput SNP typing could be readily realized by coupling ALM-ASA with a well-developed automation device for sample processing.     

Asymmetric overlap extension PCR method bypassing intermediate purification and the amplification of wild-type template in site-directed mutagenesis

By combining asymmetric PCR and overlap extension, we developed a novel asymmetric overlap extension PCR (AOE-PCR) method for site-directed mutagenesis which bypassed the need for intermediate purification and excluded the amplification of a wild-type template. This method was used to introduce single base mutations into a small GTPase gene from cotton and to simultaneously introduce two mutations just by repeating this method using the first round AOE-PCR products as template. Our results suggested that the AOE-PCR method represents a valuable improvement of the original overlap extension PCR for site-directed mutagenesis.