note the deficiency of using protein sequences since, due to the degenerate code, they
do not directly contain the whole evolutionary history of their genes. They may not
have seriously considered nucleic acid sequences because, at that time, such
sequences could not be obtained.
That all changed the same year. Frederick Sanger published a report in 1965 of a
method to sequence “fingerprints” of rRNAs from Escherichia coli and yeast
(Sanger et al. 1965). Stable RNAs extracted from cells were digested with ribonuclease T1 and the resulting oligonucleotides resolved by paper electrophoresis.
These oligonucleotides could then be extracted from the paper and digested with
ribonuclease and phosphodiesterases and the digests again resolved by paper electrophoresis. From the migrations of these products, the sequences of the oligonucleotides could be determined. Although a sequence of the entire rRNA molecules
could not be determined, it did provide a “fingerprint” of the molecule that allowed
comparisons between molecules to be made. The method also provided the basis for
further technological development of sequencing methods. Despite the fact that
DNA would contain the information most useful to evolution studies, DNA sequencing was out of the question given the size of chromosomal DNA molecules.
Interest in using protein sequences for phylogenetic analyses diminished once
nucleic acid sequencing began and proved superior for discerning evolutionary
relationships. Margaret Dayhoff began her sequence comparison work using protein
sequences, including those of cytochrome c. She also included ferredoxin sequences
that allowed her to include members of bacterial and eukaryotic lineages. When
nucleic acid sequencing became possible, she took up sequencing rRNAs and
tRNAs to extend her analyses and was among the first to do so (McLaughlin and
Dayhoff 1970; Schwartz and Dayhoff 1981). Dayhoff was the first to compile
protein sequences in a single place, at first a printed version of the Atlas of Protein
Sequence and Structure and later as a computer database stored on magnetic tape.
Another development necessary for future phylogenetic analyses was the creation
of algorithms to construct phylogenetic trees from sequence information. Walter
M. Fitch and Emanuel Margoliash devised such a method in 1967 (Fitch and
Margoliash 1967). Using cytochrome c sequences, they constructed a phylogenetic
tree that largely agreed with eukaryotic phylogenies. The only microbes included in
their analysis were yeast species and those diverged from one another in a lineage at
the base of the eukaryotic tree, as was expected. No bacteria were analyzed. This
development was important in providing an objective, quantitative way to compare
characters, in this case semantide sequences, replacing the examination by eye of
characters (morphologies or biochemical characters) of earlier phylogenetic efforts.
Dayhoff was among the first to publish phylogenetic trees constructed using
molecular sequences (Dayhoff 1969). Using composite trees that combined comparisons of the sequences of several different kinds of macromolecules, she was able
to confirm the evolutionary relationships among a small group of bacteria and the
relationship of chloroplasts and mitochondria to those bacterial lineages (Schwartz
and Dayhoff 1978). Later, after the discovery of archaebacteria, these could also be
included in her analyses. Her analysis of those indicated that the two archaebacteria
that she included, Halobacterium and Thermoplasma, were more closely related to
the cytoplasmic line of eukaryotic sequences and so did not define a separate
2 Darwin’s Science’s Impact on the Evolution of the Microbiological Sciences
39
do not directly contain the whole evolutionary history of their genes. They may not
have seriously considered nucleic acid sequences because, at that time, such
sequences could not be obtained.
That all changed the same year. Frederick Sanger published a report in 1965 of a
method to sequence “fingerprints” of rRNAs from Escherichia coli and yeast
(Sanger et al. 1965). Stable RNAs extracted from cells were digested with ribonuclease T1 and the resulting oligonucleotides resolved by paper electrophoresis.
These oligonucleotides could then be extracted from the paper and digested with
ribonuclease and phosphodiesterases and the digests again resolved by paper electrophoresis. From the migrations of these products, the sequences of the oligonucleotides could be determined. Although a sequence of the entire rRNA molecules
could not be determined, it did provide a “fingerprint” of the molecule that allowed
comparisons between molecules to be made. The method also provided the basis for
further technological development of sequencing methods. Despite the fact that
DNA would contain the information most useful to evolution studies, DNA sequencing was out of the question given the size of chromosomal DNA molecules.
Interest in using protein sequences for phylogenetic analyses diminished once
nucleic acid sequencing began and proved superior for discerning evolutionary
relationships. Margaret Dayhoff began her sequence comparison work using protein
sequences, including those of cytochrome c. She also included ferredoxin sequences
that allowed her to include members of bacterial and eukaryotic lineages. When
nucleic acid sequencing became possible, she took up sequencing rRNAs and
tRNAs to extend her analyses and was among the first to do so (McLaughlin and
Dayhoff 1970; Schwartz and Dayhoff 1981). Dayhoff was the first to compile
protein sequences in a single place, at first a printed version of the Atlas of Protein
Sequence and Structure and later as a computer database stored on magnetic tape.
Another development necessary for future phylogenetic analyses was the creation
of algorithms to construct phylogenetic trees from sequence information. Walter
M. Fitch and Emanuel Margoliash devised such a method in 1967 (Fitch and
Margoliash 1967). Using cytochrome c sequences, they constructed a phylogenetic
tree that largely agreed with eukaryotic phylogenies. The only microbes included in
their analysis were yeast species and those diverged from one another in a lineage at
the base of the eukaryotic tree, as was expected. No bacteria were analyzed. This
development was important in providing an objective, quantitative way to compare
characters, in this case semantide sequences, replacing the examination by eye of
characters (morphologies or biochemical characters) of earlier phylogenetic efforts.
Dayhoff was among the first to publish phylogenetic trees constructed using
molecular sequences (Dayhoff 1969). Using composite trees that combined comparisons of the sequences of several different kinds of macromolecules, she was able
to confirm the evolutionary relationships among a small group of bacteria and the
relationship of chloroplasts and mitochondria to those bacterial lineages (Schwartz
and Dayhoff 1978). Later, after the discovery of archaebacteria, these could also be
included in her analyses. Her analysis of those indicated that the two archaebacteria
that she included, Halobacterium and Thermoplasma, were more closely related to
the cytoplasmic line of eukaryotic sequences and so did not define a separate
2 Darwin’s Science’s Impact on the Evolution of the Microbiological Sciences
39