Barry G. Hall

The Barlow-Hall method for in vitro evolution is a simple alternative to methods such as DNA shuffling and is particularly applicable to predicting the evolution of target genes in nature. This detailed protocol includes the procedure itself and, as Appendices, describes particularly effective protocols for electroporation and for preparation of electrocompetent cells.

Phylogenetic reconstruction based upon multiple alignments of molecular sequences is important to most branches of modern biology and is central to molecular evolution. Understanding the historical relationships among macromolecules depends upon computer programs that implement a variety of analytical methods. Because it is impossible to know those historical relationships with certainty, assessment of the accuracy of methods and the programs that implement them requires the use of programs that realistically simulate the evolution of DNA sequences. EvolveAGene 3 is a realistic coding sequence simulation program that separates mutation from selection and allows the user to set selection conditions, including variable regions of selection intensity within the sequence and variation in intensity of selection over branches. Variation includes base substitutions, insertions, and deletions. To the best of my knowledge, it is the only program available that simulates the evolution of intact coding sequences. Output includes the true tree and true alignments of the resulting coding sequence and corresponding protein sequences. A log file reports the frequencies of each kind of base substitution, the ratio of transition to transversion substitutions, the ratio of indel to base substitution mutations, and the numbers of silent and amino acid replacement mutations. The realism of the data sets has been assessed by comparing the d_N/d_S ratio, the ratio of transition to transversion substitutions, and the ratio of indel to base substitution mutations of the simulated data sets with those parameters of real data sets from the “gold standard” BaliBase collection of structural alignments. Results show that the data sets produced by EvolveAGene 3 are very similar to real data sets, and EvolveAGene 3 is therefore a realistic simulation program that can be used to evaluate a variety of programs and methods in molecular evolution.

EvolveAGene 3 is a realistic coding sequence simulation program that separates mutation from selection and allows the user to set selection conditions, including variable regions of selection intensity within the sequence and variation in intensity of selection over branches. Variation includes base substitutions, insertions and deletions. Output includes a log file, the true tree and both unaligned coding sequence and protein sequences and the true DNA and protein alignments.

There are a variety of reasons to reconstruct the sequences of ancient proteins, but whatever the reason, the value of the reconstructed protein depends on the accuracy with which the ancient sequence is inferred. This study uses sequences simulated by a sequence-evolution simulation program that compares parsimony, maximum likelihood, and the Bayesian methods of inferring ancestral sequences and concludes that the Bayesian method, as implemented by mrbayes 3.11 , is preferred. Estimated ancestral sequences are of necessity the same length as the alignment on which the underlying phylogeny is based. A highly accurate method for correcting the estimated sequences is introduced, and it is shown that the correction permits inferring the sequences of ancient protein sequences with a very high degree of accuracy.

The metallo-β-lactamases fall into two groups: Ambler class B subgroups B1 and B2 and Ambler class B subgroup B3. The two groups are so distantly related that there is no detectable sequence homology between members of the two different groups, but homology is clearly detectable at the protein structure level. The multiple structure alignment program MAPS has been used to align the structures of eight metallo-β-lactamases and five structurally homologous proteins from the metallo-β-lactamase superfamily, and that alignment has been used to construct a phylogenetic tree of the metallo-β-lactamases. The presence of genes from Eubacteria , Archaebacteria , and Eukaryota on that tree is consistent with a very ancient origin of the metallo-β-lactamase family.

1Biology Department, University of Rochester, Rochester, NY, USA; 2Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA, U

A biologically realistic method was used to simulate evolutionary trees. The method uses a real DNA coding sequence as the starting point, simulates mutation according to the mutational spectrum of Escherichia coli—including base substitutions, insertions, and deletions—and separates the processes of mutation and selection. Trees of 8, 16, 32, and 64 taxa were simulated with average branch lengths of 50, 100, 150, 200, and 250 changes per branch. The resulting sequences were aligned with ClustalX, and trees were estimated by Neighbor Joining, Parsimony, Maximum Likelihood, and Bayesian methods from both DNA sequences and the corresponding protein sequences. The estimated trees were compared with the true trees, and both topological and branch length accuracies were scored. Over the variety of conditions tested, Bayesian trees estimated from DNA sequences that had been aligned according to the alignment of the corresponding protein sequences were the most accurate, followed by Maximum Likelihood trees estimated from DNA sequences and Parsimony trees estimated from protein sequences.

The metallo-β-lactamases constitute Class B in the Ambler classification of β-lactamases and are divided into three subclasses: Bl, B2, and B3. Bayesian phylogenies of the Subclass B1+B2 and Subclass B3 metallo-β-lactamases and their homologs show that the β-lactam-hydrolyzing function evolved independently within each group. In Subclass B1+B2 that function evolved about 1 billion years ago, and in Subclass B3 it evolved before the divergence of the Gram-positive and Gram-negative eubacteria, about 2 billion years ago. These results lend additional support to the proposal that the metallo-β-lactamases should be divided into two distinct classes.