Third Codon Position Research Articles

Let's keep the APGAR score in perspective

The complete mitochondrial cytochromebgenes of 53 genera of oscine passerine birds representing the major groups of finches and some allies were compared. Phylogenetic trees resulting from three levels of character partition removal (no data removed, transitions at third positions of codons removed, and all transitions removed [transversion parsimony]) were generally concordant, and all supported several basic statements regarding relationships of finches and finch-like birds, including: (1) larks (Alaudidae) show no close relationship to any finch group; (2)Peucedramus(olive warbler) is phylogenetically far removed from true wood warblers; (3) a clade consisting of fringillids, passerids, motacillids, and emberizids is supported, and this clade is characterized by evolution of a vestigial 10th wing primary; and (4) Hawaiian honeycreepers are derived from within the cardueline finches. Excluding transition substitutions at third positions of codons resulted in phylogenetic trees similar to, but with greater bootstrap nodal support than, trees derived using either all data (equally weighted) or transversion parsimony. Relative to the shortest trees obtained using all data, the topologies obtained after elimination of third-position transitions showed only slight increases in realized treelength and homoplasy. These increases were negligable compared to increases in overall nodal support; therefore, this partition removal scheme may enhance recovery of deep phylogenetic signal in protein-coding DNA datasets.

Molecular systematics of the Gelidiales: inferences from separate and combined analyses of plastid rbcL and nuclear SSU gene sequences

Nucleotide gene sequences for both the nuclear-encoded small-subunit RNA (SSU) and plastid-encoded large subunit of RuBisCO (rbcL) were determined for 16 species classified in the red algal order Gelidiales. Sequence comparisons indicate that rbcL is evolving at a faster rate than SSU in these species and that there is a more even distribution of changes across the length of the rbcL gene compared with that observed for the SSU gene. The rbcL sequences also showed a marked anti-GC bias at synonymous third-codon base positions. Parsimony and maximum-likelihood analyses were used to generate phylogenetic trees from both separate and combined analyses of the two sequence data sets. Relationships among most taxa are resolved robustly; however, the relative order of branching for the Capreolia and Ptilophora clades remains uncertain. The molecular data provide unambiguous, independent support for recognition of the newly established genus Pterocladiella. Although the position of Pterocladiella within the Gelidiales was not resolved clearly by the rbcL data alone, analyses of both the SSU and combined data matrices indicate that this genus is one of the three earliest-diverging lineages within the order. These data also suggest that Gelidium as currently circumscribed is not monophyletic. Molecular and morphological evidence suggests that the origin and, perhaps, diversification of the major lineages of gelidialean algae is correlated with distinct, independently evolved nutritional strategies for the developing carposporophyte.

Mutations at codon 249 of p53 gene in human hepatocellular carcinomas from Tongan, China

Codon 249 (exon 7) of the putative tumor suppressor gene p53 is a mutational hot-spot for hepatocellular carcinoma (HCC) but not other tumors. DNA samples from primary HCC patients from Tongan, an area of high HCC incidence in China (>40 per 100 000 population), were analyzed for specific mutations in codon 249 of the p53 gene using polymerase chain reaction (PCR)/restriction-digest methods and direct DNA sequencing. Seven of the 21 samples screened were found to have a point mutation at the third base position of codon 249 (AGG to AGT). The result is consistent with previous reports that the G→T transversion is positively associated with the level of dietary aflatoxin B 1 (AFB 1) contamination, which has been implicated as one of the risk factors in Tongan area. Of the 7 HCC patients that contained the codon 249 point mutation, one was hepatitis B virus (HBV)-negative. This is only the second documentation of an HCC patient harboring the p53 codon 249 mutation, who was HBV-negative.

Sequence variation within a nonstructural region of the hepatitis G virus genome

Nine sets of nested PCR primers from a 2.6-kb region of the hepatitis G virus (HGV) genome at nucleotide positions 5829 to 8421 were designed and used to analyze serum specimens obtained from patients with community-acquired non-A, non-B hepatitis who were HGV RNA positive. One set of primers was found to be most efficient in detecting HGV and was subsequently used to test 162 HCV-positive and 11 HCV-negative plasma units obtained from individual paid donors. HGV RNA was detected in 30 (17.3%) plasma units, 2 of which were found among the 11 HCV-negative specimens. A complete set of nine PCR fragments was obtained from two patients with community-acquired acute non-A, non-B hepatitis and from four paid donors. All PCR fragments were sequenced and were shown to have a nucleotide similarity of 85.9 to 92.3% and a derived amino acid similarity of 96.0 to 99.0%. The majority of nucleotide changes occurred in the third position of codons. The HGV nucleotide and protein sequences obtained in this study were compared with HCV sequences. Based on this analysis the 2.6-kb fragment was predicted to encode the C-terminal part of the putative NS4b, the entire NS5a, and almost the complete NS5b proteins. Putative protease cleavage sites separating these proteins were also predicted. In serial specimens obtained from the two HGV-infected patients, no significant variations were found in the HGV nucleotide and derived amino acid sequences over time. The HGV sequences obtained from one patient showed no changes over 6 months, whereas more than 99.0% homology was observed for sequences from the second patient over 2.5 years. Heterogeneity analysis performed on 10 sequences obtained in this study and corresponding regions from 6 known full-size sequences of the HGV genomes demonstrated notable discrete heterogeneity consistent with the existence of HGV genetic groups or types.

Random sequence analysis of genomic DNA of a hyperthermophile: Aquifex pyrophilus

Aquifex pyrophilus is one of the hyperthermophilic bacteria that can grow at temperatures up to 95 degrees C. To obtain information about its genomic structure, random sequencing was performed on plasmid libraries containing 0.5-2 kb genomic DNA fragments of A. pyrophilus. Comparison of the obtained sequence tags with known proteins revealed that 123 tags showed strong similarity to previously identified proteins in the PIR or Genebank databases. These included three proteases, two amino acid racemases, and three enzymes utilizing oxygen as substrate. Although the GC ratio of the genome is about 40%, the codon usage of A. pyrophilus showed biased occurrence of G and C at the third position of codons, especially those for amino acids such as asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, and tyrosine. A higher ratio of positively charged amino acids in A. pyrophilus proteins as compared with proteins from mesophiles suggested that Aquifex proteins might contain increased ion-pair interaction that could help to maintain heat stability.

Correlation of Haplotypes of a Fungal Plant Pathogen with their Respective Host Species of Origin.

The anther smut fungus, Microbotryum violaceum, infects over 200 species of Caryophyllaceae (Pinks). However, limited published studies, as well as anecdotal evidence, suggest that each isolate of the fungus is restricted to one or a few species that it can productively infect. In the absence of physical differences, it would be useful to have molecular markers to identify individuals with specific host ranges prior to genetic analyses of host preference. With this purpose in mind, 17 isolates from eight different host species were characterized for differences in their respective γ-tubulin genes. The region of the gene including the sixth and seventh introns and some surrounding coding regions was amplified and sequenced and the results were analyzed phylogenetically. Despite the small sample size and the geographical distribution of their respective host plants, isolates from the same host species showed no differences in the DNA regions examined; isolates of closely related pathovars also grouped together. In contrast, relative to the corresponding regions from other pathovars, isolates from host species that were genetically or taxonomically more distant showed a marked number of differences in both introns and in the third (wobble) position of codons in the seventh exon. Thus, DNA sequence differences in this highly conserved gene may be used to distinguish isolates from different host species. Such information may prove useful as markers for the different formae speciales in future analyses of host preference.

Codon usage patterns in Trichomonas vaginalis

Codon usage variation was studied in the parabasilid protozoan Trichomonas vaginalis. Correspondence analysis was employed to identify the single largest source of variation in the dataset. The G+C content at the third position of synonymously variable codons was also evaluated. A strong positive correlation was observed between the effective number of codons measure and the position of the genes on the axis of greatest dispersion following correspondence analysis. The correlation between base composition at the third position and effective number of codons was weaker. The results indicate that the major influence on codon usage bias is translational selection, with a moderate effect attributable to mutational pressure. In addition, twenty codons were identified whose use in biased genes is statistically significantly greater than in unbiased genes.

Codon usage in Entamoeba histolytica, E. dispar and E. invadens

We analyzed the frequencies of genetic codon usage of 68 non-redundant protein coding genes from the human-pathogenic E. histolytica (28 117 codons), 6 from the non-pathogenic E. dispar (1744 codons), and 4 from the reptilian E. invadens (933 codons). The A + U contents of the protein coding sequences from E. histolytica, E. dispar, and E. invadens were 67%, 66%, and 58%, respectively. The nucleotide frequency in the third position was strongly biased toward A + U in E. histolytica and E. dispar (85% and 82%, respectively); the degree of the A + U bias was higher in the third position than that in the first or second position. In contrast, the nucleotide frequency in the third position was less biased in E. invadens (60% A + U) than in E. histolytica and E. dispar. Codon usage was biased in accordance with the A + U preference in the third position in E. histolytica and E. dispar. However, no apparent difference in the codon usage was found between E. histolytica and E. dispar. The codon usage in E. invadens was found less biased; the nucleotide biases observed in the third position of the synonymous codons for several amino acids including leucine, tyrosine, cysteine, and histidine of the E. histolytica and E. dispar genes were reversed or absent. The codon usage in Entamoeba species significantly differed from that in other amitochondrial protist, Giardia lamblia and Trichomonas vaginalis. Two sequences encoding ribosomal protein S10 and S27 showed significantly smaller codon biases than the rest of E. histolytica sequences, suggesting that these ribosomal proteins might be under specific functional constraint of codon usage. The differences in the A + U content of the coding sequences and in the codon usage between the mammalian E. histolytica and E. dispar and the reptilian E. invadens suggested that the reptilian Entamoeba species were distantly related to the mammalian species. These results may aid in elucidating pressures that facilitate changes in the patterns of the genetic codon usage.

A novel polymorphism in the coding region of the vasopressin type 2 receptor gene.

Nephrogenic diabetes insipidus (NDI) is a rare disease characterized by renal inability to respond properly to arginine vasopressin due to mutations in the vasopressin type 2 receptor (V2(R)) gene in affected kindreds. In most kindreds thus far reported, the mode of inheritance follows an X chromosome-linked recessive pattern although autosomal-dominant and autosomal-recessive modes of inheritance have also been described. Studies demonstrating mutations in the V2(R) gene in affected kindreds that modify the receptor structure, resulting in a dys- or nonfunctional receptor have been described, but phenotypically indistinguishable NDI patients with a structurally normal V2(R) gene have also been reported. In the present study, we analyzed exon 3 of the V2(R) gene in 20 unrelated individuals by direct sequencing. A C-->T alteration in the third position of codon 331 (AGC-->AGT), which did not alter the encoded amino acid, was found in nine individuals, including two unrelated patients with NDI. Taken together, these observations emphasize the molecular heterogeneity of a phenotypically homogeneous syndrome.

Clinical, biochemical, and molecular investigations of a genetic isolate of growth hormone insensitivity (Laron's syndrome).

We have characterized the GH receptor mutation that is responsible for extreme short stature and GH insensitivity in a Bahamian genetic isolate. Heights of affected individuals ranged from -4.0 to -6.3 SD. Like others with Laron's syndrome, they had normal to high serum GH concentrations and low serum insulin-like growth factor I concentrations. Circulating levels of GH-binding protein activity were below limits of detection. Amplification of exons 2-7 and screening with single strand conformational polymorphism analysis located an abnormality in exon 7. Sequencing identified homozygosity for a C to T transition in the third position of codon 236. Reverse transcription and PCR amplification of complementary DNA from lymphocytes showed that this same sense mutation generated a new splice donor site 63 bp 5' to the normal exon 7 splice site. This novel site was used to the exclusion of the normal site in homozygotes. Both normal and variant messenger ribonucleic acid species were detected in heterozygotes. The predicted protein lacks 21 amino acids, including those defining the WS-like motif of the GH receptor extracellular domain. The high frequency of Laron's syndrome in this isolated island population probably reflects the introduction of the G236 splice mutation by a settler early in the 300-yr history of English settlement.

Evolution of the mitochondrial cytochrome oxidase II gene in collembola.

The sequence of the mitochondrial COII gene has been widely used to estimate phylogenetic relationships at different taxomonic levels across insects. We investigated the molecular evolution of the COII gene and its usefulness for reconstructing phylogenetic relationships within and among four collembolan families. The collembolan COII gene showed the lowest A + T content of all insects so far examined, confirming that the well-known A + T bias in insect mitochondrial genes tends to increase from the basal to apical orders. Fifty-seven percent of all nucleotide positions were variable and most of the third codon positions appeared free to vary. Values of genetic distance between congeneric species and between families were remarkably high; in some cases the latter were higher than divergence values between other orders of insects. The remarkably high divergence levels observed here provide evidence that collembolan taxa are quite old; divergence levels among collembolan families equaled or exceeded divergences among pterygote insect orders. Once the saturated third-codon positions (which violated stationarity of base frequencies) were removed, the COII sequences contained phylogenetic information, but the extent of that information was overestimated by parsimony methods relative to likelihood methods. In the phylogenetic analysis, consistent statistical support was obtained for the monophyly of all four genera examined, but relationships among genera/families were not well supported. Within the genus Orchesella, relationships were well resolved and agreed with allozyme data. Within the genus Isotomurus, although three pairs of populations were consistently identified, these appeared to have arisen in a burst of evolution from an earlier ancestor. Isotomurus italicus always appeared as basal and I. palustris appeared to harbor a cryptic species, corroborating allozyme data.

The complete nucleotide sequence of rabbit haemorrhagic disease virus (Czech strain V351): use of the polymerase chain reaction to detect replication in Australian vertebrates and analysis of viral population sequence variation

The complete nucleotide sequence of the Czech strain of rabbit haemorrhagic disease virus (RHDV) was determined to be 7437 nucleotides in length with a 5′-terminal non-coding region of 9 nucleotides and a 3′-terminal non-coding region of 59 nucleotides. Two open reading frames (ORFs) were found within this sequence coding for polypeptides of 2344 (nucleotides 10–7044) and 117 amino acids (nucleotides 7025–7378). The sequence of this isolate was approximately 1% different from that reported by Meyers et al., having 78 nucleotide changes which resulted in 30 amino acid differences, the majority of these clustering in the N-terminus of the large ORF and the middle of the viral coat protein. Only a single conservative amino acid change was seen in the smaller 3′-terminal ORF. Since the virus cannot at present be propagated in tissue culture, but isolated only after replication in rabbits, the reported sequence must be considered as a consensus sequence from the viral population. To gain some understanding of the possible sequence diversity within this virus population, 97 clones were sequenced from a polymerase chain reaction (PCR) fragment to determine the sequence diversity of the virus population. Four major classes of variant were described with mutations generally in the third base position of codons. A nested reverse transcriptase (RT)-PCR (using sequence derived for the coat protein of RHDV) was used to determine the presence or absence of RHDV inoculated into non-host animal species. No replication of the virus was detected in 28 different vertebrate species other than rabbits. PCR tests on both mosquitoes and fleas feeding on RHDV infected rabbits were positive. The RT-PCR test was more sensitive when compared with an antigen capture ELISA to detect the presence of genomic RNA/or virus in infected rabbits.

A Mitochondrial CytochromeBPhylogeny of theAlectorisPartridges

Nucleotide sequences of the entire mitochondrial cytochromebgene were used to study patterns of molecular evolution and phylogenetic relationships of the seven extant species ofAlectorispartridges. AmongAlectoris,transition substitutions were not saturated and 80% of nucleotide variability and informative parsimony substitutions were at third positions of codons. Transitions contributed most of interspecific genetic divergence. Phylogenetic analyses of the mtDNA sequences supported the existance of three main evolutionary events and were concordant with relationships reconstructed using allozyme data. The first splitting separated north AfricanA. barbaraand ArabianA. melanocephalafrom the otherAlectoris.Following, westernA. rufaandA. graecaseparated from eastern species. Recent speciation events originatedA. chukar, A. magna,andA. philbyi.A tentative calibration of rates of nucleotide evolution suggests thatAlectorisspeciated between ca. 6 and 2 million years ago. Consistent resolution of relationships among recent species ofAlectorismay require the addition of sequences from genes evolving faster than cytochromeb.

Evolutionary genetics of the suiformes as reconstructed using mtDNA sequencing

We have amplified and sequnced the entire mitochondrial DNA cytochromeb gene from four species of Suidae: babirusa, warthog, bearded pig, and some specimens belonging to different subspecies and populations of wild and domestic pigs (Sus scrofa). These sequences were aligned with additional mammalian sequences retrieved from the literature and were used to obtain phylogenetic trees of the Suiformes (Artiodactyla). Several species of Carnivora, Perissodactyla. Cetacea, and other Artiodactyla were used as outgroups. Molecular phylogenetic relationships among the Suiformes reflect their current taxonomy: Hippopotamidae, Tayassuidae, and Suidae are separated by deep genetic gaps, and the division of the Suidae into the subfamilies Babyrousinae., Phacochoerinae, and Suinae has strong genetic correlates. Cytochromeb sequences show differences among Asian and Western populations ofSus scrofa, agreeing with other genetic information (karyotypes blood groups, and protein variability). The two Italian subspecies of wild boar have unique mtDNA cytochromeb haplotypes. The evolutionary rates of cytochromeb sequences are different at transitions versus transversions as well as at first, second, and third positions of codons. Therefore, these classes of substitutions reached different levels of mutational saturation. Only transversions and the conservative first and second position substitutions are linearly related to genetic distances among the Suiformes. Therefore, divergence times were computed using unsaturated conserved nucleotide substitutions and calibrated using paleontological divergence times between some Artiodactyla. Transversions apparently evolve at remarkably regular rates in ungulate taxa which have accumulated less than 20% estimated sequence divergence, corresponding to about 40–45 million years of independent evolution. Molecular, information suggests that Hippopotamidae and Tayassuidae are not closely related (as stated by Pickford, 1986, 1989, 1993) and that the origin of babirusa and warthog (about 10–19 and 5–15 million years ago, respectively) is more recent than supported by current evolutionary reconstructions. The inferred origin of bearded pig is about 2.1 million years old, and genetic divergence among differentSus scrofa populations is probably a Pleistocene event. The addition of new sequences of Suiformes does not help in resolving the phylogenetic position ofHippopotamus amphibius, which shows weak but recurrent linkages with the cetacean evolutionary lineage.

A Phylogeny of the Bears (Ursidae) Inferred from Complete Sequences of Three Mitochondrial Genes

Complete sequences of DNA are described for the cytochrome b, tRNAThrand tRNAProgenes of mitochondria of four extant species of ursids and compared to sequences of four other species of ursids previously studied by us. Phylogenetic analyses indicate that the giant panda and the spectacled bear are the basal taxa of the ursid radiation. The ursines, a group which includes the sun bear, sloth bear, American black bear, Asiatic black bear, brown bear, and polar bear, experienced a rapid radiation during the mid Pliocene to early Pleistocene. The Asiatic black bear and American black bear are sister taxa. The brown bear and polar bear are the most recently derived of the ursines, with the polar bear originating from within a clade of brown bears during the Pleistocene. This paraphyletic association suggests that the rate of morphological evolution may be accelerated relative to that of molecular evolution when a new ecological niche is occupied. Calibration of the corrected average number of nucleotide differences per site with the fossil record indicates that transitions at third positions of codons in the ursid cytochrome b gene occur at a rate of approximately 6% per million years, which is considerably slower than comparable values reported for other species of mammal.

Single strand conformation polymorphism (SSCP) analysis for the detection of mutations in the CYP11B1 gene.

The adrenal 11 beta-hydroxylase is a mitochondrial P450 enzyme encoded by the CYP11B1 gene, which is situated on chromosome 8q22 in tandem with the gene for aldosterone synthase (CYP11B2). Deficiency of 11 beta-hydroxylase results in the inability to convert 11-deoxycortisol to cortisol and accounts for 5-8% of cases of congenital adrenal hyperplasia. In the following study the CYP11B1 genes from eight individuals with 11 beta-hydroxylase deficiency were screened for mutations using single strand conformation polymorphism (SSCP) analysis. Sequence analysis of variant exons revealed a 28 bp deletion and a 5 bp duplication exon 2 and five missense mutations, G267R, G267D, Q356X, R427H and C494F, distributed throughout the gene. One of these mutations, G267R, and a G to A transversion at the third nucleotide position of codon 318 occur at the +1 position of the splice donor sites. Mutations were neither the result of gene conversion nor nonhomologous recombination between the two closely related CYP11B genes.

Systematic screening for mutations in the human serotonin 1F receptor gene in patients with bipolar affective disorder and schizophrenia

Using single strand conformational analysis we screened the complete coding sequence of the serotonin 1F (5-HT1F) receptor gene for the presence of DNA sequence variation in a sample of 137 unrelated individuals including 45 schizophrenic patients, 46 bipolar patients, as well as 46 healthy controls. We detected only three rare sequence variants which are characterized by single base pair substitutions, namely a silent T-->A transversion in the third position of codon 261 (encoding isoleucine), a silent C-->T transition in the third position of codon 176 (encoding histidine), and an C-->T transition in position -78 upstream from the start codon. The lack of significant mutations in patients suffering from schizophrenia and bipolar affective disorder indicates that the 5-HT1F receptor is not commonly involved in the etiology of these diseases.

Molecular Phylogenetics and Biogeography of Tanagers in the GenusRamphocelus(Aves)

Ramphocelustanagers are distributed throughout the Neotropical lowlands. In this paper, mitochrondrial DNA (mtDNA) sequence data from cytochrome b and ND2 genes are used to estimate relationships among seven of nine species of the avian genusRamphocelus.Genetic differentiation is high betweenRamphocelus passerinii passeriniiandRamphocelus passerinii costaricensis,and the two subspecies are diagnosable and distinct from one another both morphologically and genetically. Thus, elevation to species status is recommended. Three clades are supported by both gene sequences; one clade containsR. passerinii, R. costaricensis,andR. icteronotus;the second clade containsRamphocelus carbo, Ramphocelus bresilius,andRamphocelus nigrogularis;the third clade containsRamphocelus sanguinolentus.The degree of saturation was assessed for both genes and saturation of third position of codons occurs by 10–12% uncorrected pairwise sequence divergence. The general area cladogram suggests the following area relationships: Pacific and Caribbean Central America are sister areas, Choco is the sister to the Central American area, and Amazonia/southeastern Brazil is the outgroup area to the Choco/Central American clade.

On combining protein sequences and nucleic acid sequences in phylogenetic analysis: the homeobox protein case.

Amino acid encoding genes contain character state information that may be useful for phylogenetic analysis on at least two levels. The nucleotide sequence and the translated amino acid sequences have both been employed separately as character states for cladistic studies of various taxa, including studies of the genealogy of genes in multigene families. In essence, amino acid sequences and nucleic acid sequences are two different ways of character coding the information in a gene. Silent positions in the nucleotide sequence (first or third positions in codons that can accrue change without changing the identity of the amino acid that the triplet codes for) may accrue change relatively rapidly and become saturated, losing the pattern of historical divergence. On the other hand, non-silent nucleotide alterations and their accompanying amino acid changes may evolve too slowly to reveal relationships among closely related taxa. In general, the dynamics of sequence change in silent and non-silent positions in protein coding genes result in homoplasy and lack of resolution, respectively. We suggest that the combination of nucleic acid and the translated amino acid coded character states into the same data matrix for phylogenetic analysis addresses some of the problems caused by the rapid change of silent nucleotide positions and overall slow rate of change of non-silent nucleotide positions and slowly changing amino acid positions. One major theoretical problem with this approach is the apparent non-independence of the two sources of characters. However, there are at least three possible outcomes when comparing protein coding nucleic acid sequences with their translated amino acids in a phylogenetic context on a codon by codon basis. First, the two character sets for a codon may be entirely congruent with respect to the information they convey about the relationships of a certain set of taxa. Second, one character set may display no information concerning a phylogenetic hypothesis while the other character set may impact information to a hypothesis. These two possibilities are cases of non-independence, however, we argue that congruence in such cases can be thought of as increasing the weight of the particular phylogenetic hypothesis that is supported by those characters. In the third case, the two sources of character information for a particular codon may be entirely incongruent with respect to phylogenetic hypotheses concerning the taxa examined. In this last case the two character sets are independent in that information from neither can predict the character states of the other. Examples of these possibilities are discussed and the general applicability of combining these two sources of information for protein coding genes is presented using sequences from the homeobox region of 46 homeobox genes from Drosophila melanogaster to develop a hypothesis of genealogical relationship of these genes in this large multigene family.

ON COMBINING PROTEIN SEQUENCES AND NUCLEIC ACID SEQUENCES IN PHYLOGENETIC ANALYSIS: THE HOMEOBOX PROTEIN CASE

Amino acid encoding genes contain character state information that may be useful for phylogenetic analysis on at least two levels. The nucleotide sequence and the translated amino acid sequences have both been employed separately as character states for cladistic studies of various taxa, including studies of the genealogy of genes in multigene families. In essence, amino acid sequences and nucleic acid sequences are two different ways of character coding the information in a gene. Silent positions in the nucleotide sequence (first or third positions in codons that can accrue change without changing the identity of the amino acid that the triplet codes for) may accrue change relatively rapidly and become saturated, losing the pattern of historical divergence. On the other hand, non-silent nucleotide alterations and their accompanying amino acid changes may evolve too slowly to reveal relationships among closely related taxa. In general, the dynamics of sequence change in silent and non-silent positions in protein coding genes result in homoplasy and lack of resolution, respectively. We suggest that the combination of nucleic acid and the translated amino acid coded character states into the same data matrix for phylogenetic analysis addresses some of the problems caused by the rapid change of silent nucleotide positions and overall slow rate of change of non-silent nucleotide positions and slowly changing amino acid positions. One major theoretical problem with this approach is the apparent non-independence of the two sources of characters. However, there are at least three possible outcomes when comparing protein coding nucleic acid sequences with their translated amino acids in a phylogenetic context on a codon by codon basis. First, the two character sets for a codon may be entirely congruent with respect to the information they convey about the relationships of a certain set of taxa. Second, one character set may display no information concerning a phylogenetic hypothesis while the other character set may impart information to a hypothesis. These two possibilities are cases of non-independence, however, we argue that congruence in such cases can be thought of as increasing the weight of the particular phylogenetic hypothesis that is supported by those characters. In the third case, the two sources of character information for a particular codon may be entirely incongruent with respect to phylogenetic hypotheses concerning the taxa examined. In this last case the two character sets are independent in that information from neither can predict the character states of the other. Examples of these possibilities are discussed and the general applicability of combining these two sources of information for protein coding genes is presented using sequences from the homeobox region of 46 homeobox genes from Drosophila melanogasterto develop a hypothesis of genealogical relationship of these genes in this large multigene family.