PCR, direct sequencing, and the comparative approach.
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Animals↗
Biomedical subjects
T D Kocher
Publications and source records attributed to T D Kocher.
A new antennapedia-class gene from the zebrafish.
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Amino Acid Sequence↗
Evolution of the cytochrome b gene of mammals.
With the polymerase chain reaction (PCR) and versatile primers that amplify the whole cytochrome b gene (approximately 1140 bp), we obtained 17 complete gene sequences representing three orders of hoofed mammals (ungulates) and dolphins (cetaceans). The fossil record of some ungulate lineages allowed estimation of the evolutionary rates for various components of the cytochrome b DNA and amino acid sequences. The relative rates of substitution at first, second, and third positions within codons are in the ratio 10 to 1 to at least 33. For deep divergences (greater than 5 million years) it appears that both replacements and silent transversions in this mitochondrial gene can be used for phylogenetic inference. Phylogenetic findings include the association of (1) cetaceans, artiodactyls, and perissodactyls to the exclusion of elephants and humans, (2) pronghorn and fallow deer to the exclusion of bovids (i.e., cow, sheep, and goat), (3) sheep and goat to the exclusion of other pecorans (i.e., cow, giraffe, deer, and pronghorn), and (4) advanced ruminants to the exclusion of the chevrotain and other artiodactyls. Comparisons of these cytochrome b sequences support current structure-function models for this membrane-spanning protein. That part of the outer surface which includes the Qo redox center is more constrained than the remainder of the molecule, namely, the transmembrane segments and the surface that protrudes into the mitochondrial matrix. Many of the amino acid replacements within the transmembrane segments are exchanges between hydrophobic residues (especially leucine, isoleucine, and valine). Replacement changes at first and second positions of codons approximate a negative binomial distribution, similar to other protein-coding sequences. At four-fold degenerate positions of codons, the nucleotide substitutions approximate a Poisson distribution, implying that the underlying mutational spectrum is random with respect to position.
Amino Acid Sequence↗
Comparison of mitochondrial DNA sequences of seven morphospecies of black flies (Diptera: Simuliidae).
Universal primers constructed from the 16S ribosomal RNA gene in the Drosophila yakuba mitochondrial genome were successfully used to amplify, via the polymerase chain reaction, the homologous region of mitochondrial DNA from seven black fly morphospecies. Amplification was achieved from single larval salivary glands and from single adults preserved in Carnoy's fixative (ethanol - acetic acid, 3:1), allowing DNA sequences and polytene chromosome banding pattern data to be gathered from the same individuals. Nucleotide sequences of the amplified DNA segment (347 base pairs) were obtained from all the species examined. As in Drosophila, the nucleotide base composition of the sequenced segment from black flies had a high adenine (A) and thymine (T) content (A + T on average comprised 77% of all nucleotides.). Nucleotide differences among the seven species were observed at 59 positions (55 nucleotide substitutions and 4 deletions). There were more transversion differences than transition differences both among and within genera; the proportion of transversions was higher between genera than within genera. Most transversion differences were A----T type, comprising 79% of all transversion differences and 50% of all sequence differences. Phylogenetic inference based strictly on transversion differences confirmed traditional generic and tribal groupings, i.e., Prosimulium fuscum (Syme & Davies) is close to Prosimulium magnum (Dyar & Shannon); Simulium decorum (Walker), Simulium venustum s.l. (Say), and Simulium vittatum s.l. (Zetterstedt) are close to each other; Stegopterna mutata (Malloch) and Cnephia dacotensis (Dyar & Shannon), which belong to the tribe Cnephiini, are grouped together.
Animals↗
Monophyletic origin of Lake Victoria cichlid fishes suggested by mitochondrial DNA sequences.
Lake Victoria, together with its satellite lakes, harbours roughly 200 endemic forms of cichlid fishes that are classified as 'haplochromines' and yet the lake system is less than a million years old. This 'flock' has attracted attention because of the possibility that it evolved within the lake from one ancestral species and that biologists are thus presented with a case of explosive evolution. Within the past decade, however, morphology has increasingly emphasized the view that the flock may be polyphyletic. We sequenced up to 803 base pairs of mitochondrial DNA from 14 representative Victorian species and 23 additional African species. The flock seems to be monophyletic, and is more akin to that from Lake Malawi than to species from Lake Tanganyika; in addition, it contains less genetic variation than does the human species, and there is virtually no sharing of mitochondrial DNA types among species. These results confirm that the founding event was recent.
Africa, Eastern↗
Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers.
With a standard set of primers directed toward conserved regions, we have used the polymerase chain reaction to amplify homologous segments of mtDNA from more than 100 animal species, including mammals, birds, amphibians, fishes, and some invertebrates. Amplification and direct sequencing were possible using unpurified mtDNA from nanogram samples of fresh specimens and microgram amounts of tissues preserved for months in alcohol or decades in the dry state. The bird and fish sequences evolve with the same strong bias toward transitions that holds for mammals. However, because the light strand of birds is deficient in thymine, thymine to cytosine transitions are less common than in other taxa. Amino acid replacement in a segment of the cytochrome b gene is faster in mammals and birds than in fishes and the pattern of replacements fits the structural hypothesis for cytochrome b. The unexpectedly wide taxonomic utility of these primers offers opportunities for phylogenetic and population research.
Amino Acid Sequence↗
Mitochondrial DNA sequences in single hairs from a southern African population.
Hypervariable parts of mitochondrial DNA (mtDNA) were amplified enzymatically and sequenced directly by using genomic DNA from single plucked human hairs. This method has been applied to study mtDNA sequence variation among 15 members of the !Kung population. A genealogical tree relating these aboriginal, Khoisan-speaking southern Africans to 68 other humans and to one chimpanzee has the deepest branches occurring amongst the !Kung, a result consistent with an African origin of human mtDNA. Fifteen cases of unrelated individuals having identical sequences in the most variable parts of the mtDNA control region were found within populations of !Kung, Western Pygmies, and Eastern Pygmies, but no cases of identity were evident among these populations. This and other evidence of geographic structuring of the mitochondrial diversity in Africa, together with knowledge of the rate of accumulation of base changes in human mtDNA, implies that the average rate at which female lineages have moved their home bases during hunter-gatherer times could be as low as 13 meters per year. The technique of enzymatic amplification and direct sequencing applied to readily collected, highly stable biological materials such as hairs makes it possible to examine with high resolution many representatives of virtually any population.
Africa, Southern↗