Showing papers in "Molecular Biology and Evolution in 1990"

Inference of haplotypes from PCR-amplified samples of diploid populations.

Abstract: Direct sequencing of genomic DNA from diploid individuals leads to ambiguities on sequencing gels whenever there is more than one mismatching site in the sequences of the two orthologous copies of a gene. While these ambiguities cannot be resolved from a single sample without resorting to other experimental methods (such as cloning in the traditional way), population samples may be useful for inferring haplotypes. For each individual in the sample that is homozygous for the amplified sequence, there are no ambiguities in the identification of the allele’s sequence. The sequences of other alleles can be inferred by taking the remaining sequence after “subtracting off’ the sequencing ladder of each known site. Details of the algorithm for extracting allelic sequences from such data are presented here, along with some population-genetic considerations that influence the likelihood for success of the method. The algorithm also applies to the problem of inferring haplotype frequencies of closely linked restriction-site polymorphisms.

The similarity index and DNA fingerprinting.

Abstract: DNA-fingerprint similarity is being used increasingly to make inferences about levels of genetic variation within and between natural populations. It is shown that the similarity index--the average fraction of shared restriction fragments--provides upwardly biased estimates of population homozygosity but nearly unbiased estimates of the average identity-in-state for random pairs of individuals. A method is suggested for partitioning the DNA-fingerprint dissimilarity into within- and between-population components. Some simple expressions are given for the sampling variances of these estimators.

The analysis of population survey data on DNA sequence variation.

Abstract: A technique is presented for the partitioning of nucleotide diversity into within- and between-population components for the case in which multiple populations have been surveyed for restriction-site variation. This allows the estimation of an analogue of FST at the DNA level. Approximate expressions are given for the variance of these estimates resulting from nucleotide, individual, and population sampling. Application of the technique to existing studies on mitochondrial DNA in several animal species and on several nuclear genes in Drosophila indicates that the standard errors of genetic diversity estimates are usually quite large. Thus, comparative studies of nucleotide diversity need to be substantially larger than the current standards. Normally, only a very small fraction of the sampling variance is caused by sampling of individuals. Even when 20 or so restriction enzymes are employed, nucleotide sampling is a major source of error, and population sampling is often quite important. Generally, the degree of population subdivision at the nucleotide level is comparable with that at the haplotype level, but significant differences do arise as a result of inequalities in the genetic distances between haplotypes.

Limitations of the evolutionary parsimony method of phylogenetic analysis.

Abstract: Lake's evolutionary parsimony (EP) method of constructing a phylogenetic tree is primarily applied to four DNA sequences. In this method, three quantities--X, Y, and Z--that correspond to three possible unrooted trees are computed, and an invariance property of these quantities is used for choosing the best tree. However, Lake's method depends on a number of unrealistic assumptions. We therefore examined the theoretical basis of his method and reached the following conclusions: (1) When the rates of two transversional changes from a nucleotide are unequal, his invariance property breaks down. (2) Even if the rates of two transversional changes are equal, the invariance property requires some additional conditions. (3) When Kimura's two-parameter model of nucleotide substitution applies and the rate of nucleotide substitution varies greatly with branch, the EP method is generally better than the standard maximum-parsimony (MP) method in recovering the correct tree but is inferior to the neighbor-joining (NJ) and a few other distance matrix methods. (4) When the rate of nucleotide substitution is the same or nearly the same for all branches, the EP method is inferior to the MP method even if the proportion of transitional changes is high. (5) When Lake's assumptions fail, his chi2 test may identify an erroneous tree as the correct tree. This happens because the test is not for comparing different trees. (6) As long as a proper distance measure is used, the NJ method is better than the EP and MP methods whether there is a transition/transversion bias or whether there is variation in substitution rate among different nucleotide sites.

Positive Darwinian selection promotes charge profile diversity in the antigen-binding cleft of class I major-histocompatibility-complex molecules.

Abstract: Certain major-histocompatibility-complex (MHC) loci are highly polymorphic, and the mechanism of maintenance of this polymorphism remains controversial. Recent studies of the pattern of nucleotide substitution at MHC loci have produced strong evidence that this polymorphism is maintained mainly by positive Darwinian selection that operates on the antigen recognition site (ARS) of the MHC molecule. The ARS of the class I MHC consists of three subregions: (1) the binding cleft, (2) T-cell-receptor-directed residues, and (3) outward-directed residues. Here we report that the rate of nonsynonymous nucleotide substitution is much higher in the binding cleft than in the other ARS subregions. Furthermore, nonsynonymous nucleotide substitutions that result in a change of residue side-chain charge occur significantly more frequently than expected by chance. We conclude that the main target of positive selection on the class I MHC molecules is the binding cleft of the ARS and that this selection acts primarily to promote diversity among alleles with respect to the pattern of residue side-chain charges (charge profile) in the binding cleft. These results provide additional support for the hypothesis that MHC polymorphism is maintained by overdominant selection relating to antigen-binding capacity and thus to disease resistance.

Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships.

Abstract: The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five published tetrapod sequences. When the coelacanth was used as an outgroup, Lissamphibia (living amphibians) and Amniota (amniotes) were found to be statistically significant monophyletic groups. Although little resolution was obtained among the lissamphibian taxa, the amniote sequences support a sister-group relationship between birds and mammals. Portions of the 28S ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although the phylogenetic results were inconclusive. In contrast to previous studies, deletion or down-weighting of base-paired sites were found to have little effect on phylogenetic relationships. Molecular evidence for amniote relationships is reviewed, showing that three genes (beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a bird-mammal relationship, compared with one gene (histone H2B) that favors a bird-crocodilian clade. Separate analyses of four other genes (alpha-crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined analysis of all sequence data are inconclusive, in that different groups are defined in different analyses and none are strongly supported. It is suggested that until sequences become available from a broader array of taxa, the molecular evidence is best evaluated at the level of individual genes, with emphasis placed on those studies with the greatest number of taxa and sites. When this is done, a bird-mammal relationship is most strongly supported. When regarded in combination with the morphological evidence for this association, it must be considered at least as plausible as a bird-crocodilian relationship.

Chloroplast DNA evolves slowly in the palm family (Arecaceae).

Abstract: A survey of cpDNA restriction-site variation for 22 species representing five of the six subfamilies of the palm family was conducted. Phylogenetic reconstructions based on the restriction-site data are in general agreement with conventional analyses based on morphological characters. Base-substitution estimates, derived from the restriction-site data, indicate a potential 5-13-fold decrease in substitution rates within the palms relative to rate estimates for annual plant taxa. Approximately 1,000 bp of the rbcL gene (ribulose-1,5-bisphosphate carboxylase, large subunit) were sequenced from species representing two subfamilies of the palms. The complete DNA sequence data are in accord with the restriction-site data and indicate a total rate of nucleotide substitution that is about eightfold lower than that observed for annual plants.

Ribosomal RNA gene sequences and hominoid phylogeny.

Abstract: Sequences totaling 3,500 bases from the 28S rRNA gene and from one of the ribosomal internal transcribed spacers (ITS1) have been determined for human, chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus). Analyses of the rRNA alignments show (1) a clustering of substitutions in the "variable regions" of the 28S gene, (2) a 1.5-3-fold increase in divergence in the transcribed spacer over that in the exon, and (3) that human and chimpanzee are the most closely related pair, in agreement with the results of Miyamoto et al., Sibley and Ahlquist, and Caccone and Powell.

Evolutionary relationships of class II major-histocompatibility-complex genes in mammals.

Abstract: The class II MHC molecule is a heterodimer, consisting of noncovalently associated a and p chains, which are encoded by separate genes, generally designated A and B genes, respectively. The class II MHC of mammals consists of a number of

Distribution of hobo transposable elements in the genus Drosophila.

Abstract: This study describes the distribution of hobo-hybridizing sequences in the genus Drosophila. Southern blot analysis of 134 species revealed that hobo sequences are limited to the melanogaster and montium subgroups of the melanogaster-species group. Of the hobo-bearing species, only D. melanogaster and two of its sibling species, D. simulans and D. mauritiana, were found to contain potentially complete hobo elements. The distribution of hobo sequences is one of the narrowest distributions thus far described for any Drosophila transposable element.

Phylogenetic analysis of nucleoproteins suggests that human influenza A viruses emerged from a 19th-century avian ancestor.

Abstract: The nucleoprotein (NP) is one of the main determinants of species specificity of influenza A viruses. Using 25 NP sequences we have constructed evolutionary trees by the strict-parsimony procedure of Fitch (197 1). In contrast to the evolutionarygene tree, the tree based on amino acid sequences unravels remarkable differences between avian and human NPs, differences which are best explained by a strong differential selection pressure on the human NPs. It is speculated that this selection pressure is caused by a change of the host and the (T-cell) immune response. A cautious extrapolation of the tree suggests that the human influenza A virus NPs evolved 150 years ago from an avian ancestor. Furthermore, the ancestral relation between the NPs of influenza A, B, and C viruses was analyzed. The influenza A virus has a genome consisting of eight RNA segments. Its NP gene seems to play the major role in host range: (1) Rescue of NP ts mutants of fowl plague virus (H7N1, FPV) in chicken cells is possible by avian but not by human H3N2 strains. However, the formation of FPV reassortants with the NP gene of the human Hong Kong virus (H3N2) can easily be achieved in dog kidney cells. These FPV reassortants with the Hong Kong NP do not multiply in chicken cells and are nonpathogenic for chickens (Scholtissek et al. 1978, 1985). Replacement of the other FPV genes by those of the human Hong Kong virus leads to FPV reassortants that replicate well in chicken cells (Scholtissek et al. 1976). Thus, by specific replacement of the NP gene the host range has been changed. (2) This view is strengthened by the observation that the sole replacement of the NP gene of a human influenza A strain by the NP gene of an avian virus is sufficient for an efficient attenuation of the human virus for monkeys (Snyder et al. 1987). In nature, avian influenza A viruses do not spread in the human population, and human viruses do not spread in birds. In the laboratory, however, ducks can become infected artificially by human H3N2 viruses. These viruses induce relatively high antibody titers in the birds, but the viruses are not excreted. In contrast, ducks infected with duck H3N2 viruses induce only very low antibody titers and excrete these viruses (Scholtissek et al. 1985), which in this way can spread in the duck population. The NP genes-and not other genesare responsible for keeping the two large separately evolving reservoirs of influenza A viruses-that in humans and that in water birds (Hinshaw and Webster 1982; Kawaoka et al. 1988)-apart. Therefore it was of interest to search for a common ancestor of these viruses. Furthermore, we wanted to know to what extent the NPs of influenza B and C viruses were related to the NP of influenza A viruses. A thorough comparison of sequences of the NPs of many strains should give an answer. The sequences of many NP genes of influenza A viruses have been determined (Winter and Fields 198 1; Huddleston and Brownlee 1982; van Rumpuy et al. 1982;

Correcting parsimonious trees for unseen nucleotide substitutions: the effect of dense branching as exemplified by ribonuclease.

Abstract: In a study of mammalian ribonuclease evolutionary rates, we applied the FitchBruschi correction to reduce the bias caused by an unequal sampling of taxa in different lineages. The correction was clearly appropriate but only up to a point. The analysis showed that the sampling of taxa within the pecora was sufficiently intense that no correction for unseen, amino acid-changing, nucleotide substitutions was required. It was also found that the ribonuclease gene was duplicated at least twice at the origin of the pecoran branch of the artiodactyls.

Molecular characterization of a repeat element causing large-scale size variation in the mitochondrial DNA of the sea scallop Placopecten magellanicus.

Abstract: The scallop Placopecten magellanicus has the largest reported animal mitochondrial DNA (average 35 kb) and exhibits large inter- and intraindividual length variation owing to the varying copy number of a repeated element. We have characterized the repeat array by using restriction mapping and sequence analysis. The repeated element consists of 1,442 bp flanked on either side by the sequence ACTTTCC in a direct orientation. The array contains two to eight copies of the repeated element arranged in a direct orientation and in tandem. Only complete copies of the element are present in the array. The repeat element contains three regions with characteristic nucleotide sequences: a 10-bp inverted repeat shown to extrude into a cruciform in a supercoiled DNA plasmid, a 120-bp tract rich in G/C (70%) and adjacent to the inverted repeat, and periodically interspersed homopolymer runs of A and T occurring near the middle of the element which induce DNA curvature in dimeric constructs of the element. The element appears to be unique to P. magellanicus. The structural properties of the repeat element and its organization in an array of repeats may be important in explaining the generation and maintenance of large-scale mitochondrial DNA size variation observed in many animal species.

Hierarchical analysis of population genetic variation in mitochondrial and nuclear genes of Daphnia pulex.

Abstract: The geographic structure of Daphnia pulex populations from the central United States is analyzed with respect to isozyme and mitochondrial DNA variation. The species complex consists of cyclic and obligate parthenogens. A hierarchical analysis of population structure in the cyclic parthenogens by using a fixation-index approach indicates that this is one of the most extremely subdivided species yet studied. This genetic structure, much of which accrues within 100 km, is certainly due in part to the limited dispersal ability of Daphnia. However, previous work has shown that fluctuating selection can account for the spatial heterogeneity in isozyme frequencies in these populations. This may explain why the population subdivision for the mitochondrial genome increases approximately three times as rapidly with distance as does that for nuclear genes, which is slower than the neutral expectation. The obligate parthenogens are shown to be polyphyletic in origin, evolutionarily young, and, in some cases, geographically widespread.

Phylogenetic relationships of the pipid frogs Xenopus and Silurana: an integration of ribosomal DNA and morphology.

Abstract: Relationships of the pipid frog genus Silurana (= Xenopus tropicalis group of some authors) are of particular interest to developmental and molecular biologists because of the purported ancestral (i.e., unduplicated) karyotype of S. tropicalis relative to the genus Xenopus. Although most previous studies have assumed that Silurana is the sister group of Xenopus, recent morphological work suggests that Silurana is more closely related both to the South American genus Pipa and to the African genera Hymenochirus and Pseudhymenochirus than it is to Xenopus. We examined 1,486 bp of relatively variable regions of the ribosomal DNA array (including portions of the 18S and 28S genes, as well as part of an internal transcribed spacer) in Hymenochirus, Silurana, and Xenopus, as well as the outgroup genus Spea, in order to test the alternative hypotheses of relationships for Silurana. Maximum-parsimony analysis using bootstrapping and an analysis using Lake's method of invariants both significantly support the sister-group relationship between Xenopus and Silurana rather than the relationship suggested by morphology. Analysis of the combined morphological/molecular data matrix also significantly supports the Xenopus-Silurana relationship. Although our results are not inconsistent with the recognition of the genus Silurana to accommodate the species formerly called X. tropicalis and X. epitropicalis, the proposed relationships do not require the recognition of this genus in order to render Xenopus monophyletic.

Molecular analysis of duplicated esterase genes in Drosophila melanogaster.

Abstract: Genomic clones containing sequences homologous to an esterase 6 (Est-6) cDNA clone were isolated from a library of Drosophila melanogaster DNA. Comparison of the genomic and cDNA sequences revealed that the Est-6 gene comprises two exons, one of 1,387 bp and one of 248 bp, separated by a short intron of 51 bp. Further sequencing revealed the presence of a tandem duplication of the Est-6 gene (denoted Est-P) which also has an exon of 1,387 bp and an exon of 248 bp, separated by a short intron of 56 bp. The two genes show similarities of 64% and 60% at the DNA and protein levels, respectively. The coding regions of the genes are 197 bases apart, and presumptive 5' regulatory sequences of Est-P overlap at least the 3' noncoding region of Est-6. Transcripts homologous to Est-P were detected in late larvae and adults of each sex, whereas Est-6 transcripts are present in all life stages but are predominant in adult males. This suggests different physiological functions for the products of the two genes. Southern and Northern blot hybridization analyses of the 20-kb region surrounding the Est-6/Est-P duplication failed to detect any other duplicated esterase genes, although this region is actively transcribed.

Variation in the distribution of silver-staining nucleolar organizer regions on the chromosomes of the wild mouse, Mus musculus.

Abstract: A silver staining method was used to analyze the distribution of nucleolar organizer regions (Ag-NORs) on chromosomes of 45 wild mice (Mus musculus). The four subspecies represented were M. m. musculus, M. m. molossinus, M. m. castaneus, and M. m. bactrianus. Ag-NORs were observed near the centromeric regions of 11 chromosomes (4, 8, 9, 10, 11, 12, 15, 16, 17, 18, and 19), indicating a preponderance of Ag-NORs on smaller chromosomes. The first five loci have not been observed previously. It is suggested that a correlation may exist between the specific features of mouse Ag-NORs and the events involved in intra- and interchromosomal homogenization of rDNA.

Heterochromatin condensation and evolution of unique satellite-DNA families in two parasitic wasp species: Diadromus pulchellus and Eupelmus vuilleti (Hymenoptera).

Abstract: Large quantities of satellite DNA families ( 15%25% of the genome) were found in the DNA of two species of parasitic wasps, Diadromus pulchellus and Eupelmus vuilleti. In both species the satellite DNA was found to consist wholly or largely of a single family unique to that species. Several clones of each family were obtained and sequenced. Palindromes in each consensus sequence suggest the formation in vivo of hairpin structures that may play a role in the mode of heterochromatin condensation in these insects. The ancestral repeating motifs were determined from the consensus sequences. Plausible scenarios are presented for the evolution of the two satellite DNAs. The occurrence of only one family of satellite DNAs in both species may indicate that, in male haploids, such families have shorter persistence times than necessary for the origins of new duplicated sequences.

Evolution of isopenicillin N synthase genes may have involved horizontal gene transfer.

Abstract: The isopenicillin N synthase genes from three fungal species, three Gram-positive species, and one Gram-negative bacterial species share an unusually high sequence similarity. A phylogentic analysis was carried out to determine which type of evolutionary scenario best accounts for this similarity. The most plausible scenario is one in which a horizontal gene-transfer event, from the prokaryotes to the eukaryotes, occurred at a time close to the divergence between the Gram-positive and the Gram-negative bacteria.

Contrasting evolutionary histories of chloroplast thioredoxins f and m.

Abstract: Fourteen thioredoxin sequences were used to construct a minimal phylogenetic tree by using parsimony. The bacterial thioredoxins clustered into three groups: one containing the photosynthetic purple bacteria, Escherichia and Corynebacterium; a second containing the photosynthetic green bacterium, Chlorobium; and a third containing cyanobacteria. These groupings are similar to those generated from earlier 16s RNA analyses. Animal thioredoxins formed a fourth group. The two thioredoxins of chloroplasts (f and m) showed contrasting phylogenetic patterns. As predicted from prior studies, spinach chloroplast thioredoxin m grouped with its counterparts from cyanobacteria and eukaryotic algae, but, unexpectedly, thioredoxin f grouped with the animal thioredoxins. The results indicate that, during evolution, thioredoxin m of contemporary photosynthetic eukaryotic cells was derived from a prokaryotic symbiont, whereas thioredoxin f descended from an ancestral eukaryote common to plants and animals. The findings illustrate the potential of thioredoxin as a phylogenetic marker and suggest a relationship between the animal and f-type thioredoxins.

Colonization of America by Drosophila subobscura: effect of the founder event on the mitochondrial DNA polymorphism.

Abstract: The recent colonization of South America (Brncic and Budnik 1980; Brncic et al. 198 1) and North America (Beckenbach and Prevosti 1986; Prevosti et al. 1987) by the Paleartic species Drosophila subobscura has offered the opportunity to test empirically the effect of a founder event on different levels of polymorphism in an expanding population. Analysis of chromosomal inversion polymorphism (Prevosti et al. 1988), allozyme polymorphism (Prevosti et al. 1983), gametic associations of allozymes and chromosomal gene arrangements (Prevosti et al. 1983)) and allelism of lethals (Mestres et al., accepted) showed that only a mild founder effect occurred at the beginning of the colonization, but not during its expansion. Owing to its mode of inheritance, the variability of the mitochondrial DNA (mtDNA) is more sensitive to bottlenecks in population size than are nuclear genes. For this reason, it seems an ideal tool to get additional information about the colonization process. Although Latorre et al.‘s ( 1986) study of mtDNA variation in D. subobscura includes several American strains, their survey was not carried out at an intrapopulation level. In the present study, mtDNA variation has been analyzed in a total of 105 D. subobscura isofemale lines originating from North America (Eureka, 11; Gilroy, 26), South America (Santiago, 29; Valdivia, 6), and Europe (Barcelona, 33 ) . mtDNA was prepared by following a shorter version of our previous protocol (Afonso et al. 1988). The method is based on two steps of selective isolation of covalently closed circular DNA molecules, previously used for the isolation of plasmid DNA from bacteria. The first step involves alkaline denaturation (Birnboim and Doly 1979), and the second involves an acid-phenol extraction (Zasloff et al. 1978). One hundred fifty adult flies were used for its preparation, which gives enough pure mtDNA for lo12 restriction analyses. The same number of third-instar larvae gives similar yields. Flies are ground very gently in 1.75 ml precooled solution I [ 10 mM Tris, 60 mM NaCl, 5% sucrose (w/v), 10 mM EDTA (ethylenediamine tetracetate) pH = 8.01. Then 1.75 ml solution II [ 300 mM Tris, 5% sucrose (w/v), 10 mM EDTA, 1.25% sodium dodecyl sulfate ( SDS), 0.8% diethylpyrocarbonate (w/v) , freshly mixed] was added, and the solution was mixed and incubated at 65°C for 30 min. The DNA was denatured by adding 300 ~1 NaOH 2 N, so as to obtain a pH of 12.5, and was kept on ice for 6 min. SDS-potassium acetate (KAc) DNA deproteinization was performed by adding 600 ~1 KAc 5 M, pH = 5.0, and the resulting solution was mixed well and incubated at -20°C for 8 min. After centrifugation at 13,000 rpm for 10 min, the DNA-containing supematant was transferred to a 15-ml centrifuge tube. The acidphenol extraction was carried out by adding 4 ml phenol saturated with Tris-HCl (pH = 7.5), and the resulting solution was mixed by shaking and then was kept on ice for 5 min. The aqueous phase (pH 6-6.5)) was collected, and the traces of phenol were eliminated with a chloroform extraction. Finally the DNA precipitation was performed by adding 2 vol absolute ethanol. All samples were assayed for 11 restriction endonucleases: nine of them (BamHI,

Evidence for rolling-circle replication in a major satellite DNA from the South American rodents of the genus Ctenomys.

Abstract: A major PvuII satellite DNA has been cloned from a South American octodontid rodent of the genus Ctenomys (C. porteousi). The satellite monomer, termed RPCS, is 337 bp in size and 42% G + C. Analysis of the nucleotide sequence demonstrates that RPCS is not composed of a series of shorter repeats. RPCS-related sequences were found in 11 of 12 Ctenomys species analyzed by hybridization under high-stringency conditions. The only negative species, C. opimus, was reactive under low-stringency conditions. RPCS-related sequences were not found under high- or low-stringency conditions in Calomys musculinus and Mus musculus. However, under low-stringency conditions, RPCS-related sequences were found in the octodontid Octodontomys gliroides, which is thought to have diverged from the genus Ctenomys more than 10 Myr ago. The pattern of periodicities observed, by restriction analysis, between Ctenomys species in the satellite array can be mainly accounted for by a rolling-circle amplification mechanism but cannot be solely accounted for by unequal crossing-over.

Mitochondrial DNA variation and genetic structure in Old-World populations of Drosophila subobscura.

Abstract: To discover the relation between mitochondrial DNA (mtDNA) polymorphism and the geographic population structure of Drosophila subobscura previously established for other genetic traits, a wide Paleartic survey was carried out. A total of 24 nucleomorphs was observed among 261 isofemale lines assayed by 11 restriction endonucleases with 38 different sites in the mtDNA cleavage map. The differentiation of the Canary Islands populations (delta = 0.0119) compared with the mean among all the other continental and insular populations (delta = 0.0002) is striking. Both the great divergence among Canary Islands nucleomorphs (delta = 0.021) compared with the maximum nucleomorph distance in all other populations (delta = 0.017) and the abundance of endemic nucleomorphs (11) on the Canary Islands (50% of the total number of different nucleomorphs found in the entire distribution area) suggest that this molecular differentiation most probably results from the very old age of the Canary Islands populations rather than from drift and founder effects.

Cloning of the esterase-5 locus from Drosophila pseudoobscura and comparison with its homologue in D. melanogaster.

Abstract: A clone of the esterase-5 (Est-5) gene from Drosophila pseudoobscura has been isolated by hybridization to the cloned Est-6 gene of D. melanogaster. Southern analysis and sequencing of the cloned DNA revealed three regions of similarity to Est-6 that have been tentatively identified as genes, Est-5A, Est-SB, and Est-SC. Introduction of each of the three genes separately into D. melanogaster by P-element transformation has demonstrated that Est-5B encodes an enzyme with the same physical properties as EST 5 in D. pseudoobscura. Sequence analysis indicates that Est-5B encodes a 545-amino-acid protein and is composed of two exons separated by a 5.5-bp intron in the same position as the 5 I-bp intron in Est-6. Comparison of the Est-5B coding region with that of Est-6 reveals an overall similarity (73% at both the nucleotide and amino acid levels) that is substantially lower than that for other genes sequenced in both of these species. Total nucleotide and nonsynonymous site differences between Est-6 and Est-5B are more abundant in the second exon than in the first, suggesting differential effects of selection or mutation on these two exons. Comparisons of the 5'-flanking DNA of Est-SB and Est-6 reveal four short conserved sequence elements, but the remaining upstream sequences show no sig- nificant similarity. Conservation in the 3'-flanking DNA is limited to the presence of two polyadenylation sites that may correlate with the existence of two transcripts from both Est-5B and Estd. The patterns of nucleotide substitutions and amino acid replacements between Est-5B and Est-6 are consistent with the hypothesis that mutation and genetic drift are responsible for the differences between these two genes.

Single-copy DNA distance between two congeneric sea urchin species exhibiting radically different modes of development.

Abstract: We have investigated the differences between nuclear genomes of two purportedly congeneric species of sea urchin that differ radically in early development. Heliocidaris tuberculata develops by means of a typical pluteus larva, whereas H. erythrogramma develops directly from an egg that is 100-fold the volume of the H. tuberculata egg. Reassociation kinetic analysis shows that the kinetic components of the genomic DNA from the two species are essentially the same. No single repeat component explains the 30% difference between the H. erythrogramma and H. tuberculata genomes. Reciprocal hybridization of tracer-labeled single-copy DNA fractions between these species indicates that approximately 50% of the single-copy DNA is sufficiently similar to form hybrids at standard hybridization criterion. Thermal denaturation profiles of the hybridized single-copy DNA sequence yields median (T50H) values of 13.8 degrees-16.5 degrees C. This result suggests a divergence time of 10-13 Mya, which is comparable to divergence times between congeneric sea urchin species in other genera that do not differ significantly in development. Radical differences in early developmental processes can evolve rapidly between closely related forms.

Evolution of beta-globin haplotypes in human populations.

Abstract: The beta-globin haplotypes of 852 chromosomes from 12 populations in the Asia-Pacific region are described. These data are combined with those from other populations in an investigation of the affinities of regional human populations. Both partial maximum-likelihood and distance Wagner methods indicate that Africans are the most divergent group, with the remaining populations branching in the following order: Australian Aborigines, Highland Melanesians, Lowland Melanesians, Indonesians and Micronesians, Polynesians, east Asians, Indians, and Europeans. This pattern of relationship is consistent with that indicated by other data. Analysis of the evolution and distribution of haplotype occurrence provides some limited support for an origin of modern humans in Africa. Otherwise, however, it was not useful in further elucidating the evolutionary history of human populations.

Evolution of the glucose dehydrogenase gene in Drosophila.

Abstract: The glucose dehydrogenase genes (Gld) of Drosophila melanogaster, of D. pseudoobscura, and of D. virilis have been isolated and compared with each other in order to identify conserved and divergent aspects of their structure and expression. The exon/intron structure of Gld is conserved. The Gld mRNAs are similar, with a range of 2.6-2.8 kb among the three species. All three species exhibit peaks of Gld expression during every major developmental stage, although considerable variation in the precise timing of these peaks exists between species. Interspecific gene transfer experiments demonstrate that the regulation and function of the D. pseudoobscura Gld is similar enough to the homologous gene in D. melanogaster to substitute for its essential role in the eclosion process. Comparison of the putative promoter sequences has identified both shared and divergent sequence elements which are likely responsible, respectively, for the conserved and divergent patterns of expression observed. The entire coding sequences of the pseudoobscura and melanogaster Gld genes are presented and shown to encode a 612-amino-acid pre-protein. The inferred amino acid sequences are 92% conserved between the two species. In general the intronic regions of Gld are unusually well conserved.

Accuracy of estimating genetic distance between species from short sequences of mitochondrial DNA.

Abstract: Recent advances in molecular techniques allow rapid determination of genetic divergence, and in many circumstances the polymerase chain reaction (PCR) and DNA sequencing have become the technologies of choice for estimating genetic distance. For evolutionary studies, considerable effort is being focused on “universal” oligonucleotide primers that amplify short sequences of mitochondrial DNA. Kocher et al. ( 1989, p. 6 199) encapsulate this approach when they state that a “short sequence from a piece of the cytochrome b gene contains phylogenetic information extending from the infraspecific level to the intergeneric level.” If this is a valid generalization, then the advent of universal primers and the ease of direct sequencing of PCR products provides an efficient and reliable means of determining genetic divergence and inferring phylogenetic relationships among taxa. Indeed, DNA sequence derived from the cytochrome b primers (usually amounting to 250 bp) have proved successful for inferring evolutionary relationships and estimating rates and processes of molecular evolution (Kocher et al. 1989; Thomas et al. 1989). However, before evolutionary biologists turn their attention to such short DNA sequences, it is important to understand the accuracy of genetic distances estimated from such sequences. It is well known that the distribution of substitution events is leptokurtic. Thomas and Beckenbach ( 1989) showed that there is spatial heterogeneity in the distribution of substitutions in mitochondrial protein-coding genes from salmonid fishes. Nonrandom distribution of substitutions is a problem in estimating evolutionary relatedness from short, amplified DNA sequences because the sampling scheme is nonrandom, being entirely dependent on the choice of primers used for amplifications. Amplification and sequencing of fragments that are too small to encompass the scope of spatial heterogeneity in the distribution of substitutions will give biased estimates of genetic distance. * We investigated the influence of the number of basepairs on genetic distance estimates based on mtDNA sequence data by randomly subsampling known DNA sequences and then determining the variance of genetic distance estimates among subsamples. This approach allows us to investigate the extent to which substitution differences in different parts of a gene interfere with the robustness of genetic distance estimates based on short sequences. The accuracy of between-taxa genetic divergence estimation based on a small number of basepairs was evaluated by subsampling regions of the mitochondrial genomes containing protein-coding genes for five pairs of vertebrate taxa (fig. 1). The analysis focuses on fourfold-degenerate sites within protein-coding regions because these sites are free from selective constraints and therefore provide the best type of data for inferring evolutionary distance ( Wu and Li 1985 ) . For each of the five paired vertebrate comparisons, continuous, homologous sequences of 250, 500, 750, 1,000, 1,250, and 1,500 bp were selected at random from the complete sequences. For these sequence lengths, the average numbers of fourfold-

Molecular evolution of the zinc-containing long-chain alcohol dehydrogenase genes.

Abstract: Phylogenetic relationships and rates of nucleotide substitution were studied for alcohol dehydrogenase (ADH) genes by using DNA sequences from mammals and plants. Mammalian ADH sequences include the three class I genes and a class II gene from humans and one gene each from baboon, rat, and mouse. Plant sequences include two ADH genes each from maize and rice, three genes from barley, and one gene each from wheat and two dicots, Arabidopsis and pea. Phylogenetic trees show that relationships among ADH genes are generally consistent with taxonomic relationships: mammalian and plant ADH genes are classified into two distinct groups; primate class I genes are clustered; and two dicot sequences are clustered separately from monocot sequences. Accelerated evolution has been detected among the duplicated ADH genes in plants, in which synonymous substitutions occurred more often within the coenzyme-binding domain than within the catalytic domains.

The primary structure of langur (Presbytis entellus) pancreatic ribonuclease: adaptive features in digestive enzymes in mammals.

Abstract: The primary structure of pancreatic ribonuclease from langur (Presbytis entellus) has been determined. This sequence differs from that of human pancreatic ribonuclease at 14 (11%) of the amino acid positions. Eight of these 14 differences involve changes of charge, with the langur enzyme having five fewer positive charges than the human enzyme. The difference in charge between human and langur ribonuclease may be an adaptation to the different requirements for a nondigestive and a digestive role, respectively. A number of similarities in expression, gene duplications, and properties between mammalian ribonucleases and lysozymes have been observed, indicating similar adaptations in both enzyme systems.