Principles of Plant Genetics and Breeding

Analysis
BIOTECHNOLOGY IN PLANT BREEDING 241
Two evolutionary concepts underlie the inferences from analyses of multiple sequence alignments. The first is that the mutation of
one sequence residue to another is a random event in nature and that all residues are more or less equally subject to mutation. The
second is that the conservation of specific residues is maintained through evolutionary selection; that is, mutations that adversely
affect the ability of a molecule to carry out its biochemical activity or physiological role will be eliminated by reducing the ability
of the organism to live. Details of the evolutionary history between different pairs of sequences can lead to different inferences
about the properties of the sequences.
In the evolutionary history of some pairs of sequences – orthologues or orthologous sequences – the sequences have only speciation
events in their evolutionary history. Other pairs of sequences – paralogues or paralogous sequences – have one or more
gene duplication events in their common evolutionary history as well as having speciation events. In general, paralogues will
carry out the same biochemistry on different substrates or with different cofactors; while orthologues will carry out the same biochemistry
on the same substrates and will often serve an identical physiological role in the same pathways in different organisms.
Homologues include both orthologues and paralogues. Since paralogues carry out the same basic biochemistry, for example
reduce an aldehyde, the residues responsible for this activity are conserved. But, the residues responsible for the physiological
role (e.g., substrate recognition, which specific aldehyde to reduce or lipid to bind) will be under different evolutionary pressures
and will often diverge. A complete analysis of the multiple sequence alignment includes identifying residues responsible for the
common, shared properties of the entire family and the residues that discriminate between paralogous groups (Figure 1).
The results from the analysis of multiple sequence alignments is illustrated in the results obtained in the decades’ long quest to
understand how each of the 20 aminoacyl tRNA synthetase (aaRS) enzymes map each of the 60 tRNAs to its correct cognate
amino acid. The problem, as outlined by Sir Francis Crick in 1957, is where is the information that allows an aaRS to recognize
only the tRNAs encoding the correct amino acid? Experimental groups have employed a number of creative and innovative
approaches towards answering this question (Söll & Schimmel 1974). McClain, Nicholas, and coworkers applied computational
techniques, beginning with creating an accurate multiple sequence alignment of the tRNAs.
The tRNA multiple sequence alignment was divided into 20 subsets, each based on their amino acid acceptor activity (McClain
& Nicholas 1987). First these authors identified the sequence positions in the 66 tRNA sequences from Escherichia coli, its bacteriophages,
and near relatives that were conserved. They then examined the remaining positions and asked the question: what
Bpi_Bovin : VTCSSCSSHINSVHVHISKSK-VGWLIQLFSKKIESALRNKMNSQVCEKVTNSVSSKLQPYFQTLP
Bpi_Human : ITCSSCSSDIADVEVDMSGD--SGWLLNLFHNQIESKFQKVLESRICEMIQKSVSSDLQPYLQTLP
Lbp_Human : GYCLSCSSDIQNVELDIEGD--LEELLNLLQSQIDARLREVLESKICRQIEEAVTAHLQPYLQTLP
Lbp_Rabit : VTTSSCSSRIRDLELHVSGN--VGWLLNLFHNQIESKLQKVLESKICEMIQKSVTSDLQPYLQTLP
Lbp_Rat : VTASGCSNSFHKLLLHLQGEREPGWIKQLFTNFISFTLKLVLKGQICKEI-NVISNIMADFVQTRA
Cetp_Human: TDAPDCYLSFHKLLLHLQGEREPGWIKQLFTNFISFTLKLVLKGQICKEI-NIISNIMADFVQTRA
Cetp_Macfa: TDAPDCYLAFHKLLLHLQGEREPGWLKQLFTNFISFTLKLILKRQVCNEI-NTISNIMADFVQTRA
Cetp_Rabit: TNAPDCYLAFHKLLLHLQGEREPGWLKQLFTNFISFTLKLILKRQVCNEI-NTISNIMADFVQTRA
Pltp_Human: VSNVSCQASVSRMHAAFGGT--FKKVYDFLSTFITSGMRFLLNQQICPVLYHAGTVLLNSLLDYVP
Pltp_Mouse: VSNVSCEASVSKMNMAFGGT--FRRMYNFFSTFITSGMRFLLNQQICPVLYHAGTVLLNSLLDTVP
Lplc3_Rat : LILKRCNT----LLGHISLT--SGLLPTPIFGLVEQTLCKVLPGLLCPVV-DSVLSVVNELLGATL
Lplc4_Rat : LVIERCDT----LLGGIKVKLLRGLLPNLVDNLVNRVLANVLPDLLCPIV-DVVLGLVNDQLGLVD
Consensus C i l C t
Figure 1 A section of a multiple sequence alignment of 12 sequences from the Bpi/Lbp/Lp1 superfamily of
lipid-binding protein sequences. The 12 sequences belong to six different paralogous families, each of which binds a
different lipid and performs a different physiological role, generally a transport function. Each sequence is identified
by a label that identifies the paralogous family (they have a gene duplication event separating the families) before the
underscore character in the protein name and the species after the underscore. The protein names are taken from the
Swiss-Prot database. The annotation associated with each sequence in the Swiss-Prot database describes the lipid
bound and the physiological role of the protein. The sequences within each paralogous family are orthologous
(related to each other only by speciation events). The dash sequence character indicates an alignment column where
either the amino acids replaced by dashes have been lost through a deletion event or amino acids shown by the single
letter codes have been inserted into the sequence during the course of evolution. The section marked with a black
background and white letters is a moderately well-conserved motif. Such conservation often marks regions
important to the structure or function of the protein. Amino acids highlighted with black letters on a light gray
background are identical within the highlighted family of orthologous sequences and have quite different chemical
or physical properties to those in the same column in other families. They may mark positions that are responsible
for the differences among the paralogous families.