Thirty P1 clones from the X chromosome (Muller's A element) of Drosophila melanogaster were cross-hybridized in situ to Drosophila subobscura and Drosophila pseudoobscura polytene chromosomes. An additional recombinant phage lambda Dsuby was also used as a marker. Twenty-three (77%) of the P1 clones gave positive hybridization on D. pseudoobscura chromosomes but only 16 (53%) did so with those of D. subobscura. Eight P1 clones gave more than one hybridization signal on D. pseudoobscura and/or D. subobscura chromosomes. All P1 clones and lambda Dsuby hybridized on Muller's A element (X chromosome) of D. subobscura. In contrast, only 18 P1 clones and lambda Dsuby hybridized on Muller's A element (XL chromosomal arm) of D. pseudoobscura; 4 additional P1 clones hybridized on Muller's D element (XR chromosomal arm) of this species and the remaining P1 clone gave one hybridization signal on each arm of the X chromosome. This latter clone may contain one breakpoint of a pericentric inversion that may account for the interchange of genetic material between Muller's A and D elements in D. pseudoobscura. In contrast to the rare interchange of genetic material between chromosomal elements, profound differences in the order and spacing of markers were detected between D. melanogaster, D. pseudoobscura and D. subobscura. In fact, the number of chromosomal segments delimited by identical markers and conserved between pairwise comparisons is small. Therefore, extensive reorganization within Muller's A element has been produced during the divergence of the three species. Rough estimates of the number of cytologically detectable inversions contributing to differentiation of Muller's A element were obtained. The most reliable of these estimates is that obtained from the D. pseudoobscura and D. melanogaster comparison since a greater number of markers have been mapped in both species. Tentatively, one inversion breakpoint about every 200 kb has been produced and fixed during the divergence of D. pseudoobscura and D. melanogaster.

Ayala, FJ, DE Krane, and DL Hartl. 1994. “Genetic variation in IncI1-ColIb plasmids.” J Mol EvolJ Mol EvolJ Mol Evol 39: 129-33. Abstract
Nucleotide sequences of portions of three plasmid genes (cib, cir, and abi) present in IncI1-ColIb colicin plasmids obtained from strains of Salmonella typhimurium isolated in either 1974 (Barker strains) or between 1935 and 1941 (Murray strains) were examined along with sequences of the chromosomal gene for 6-phosphogluconate dehydrogenase (gnd). Our principal findings were: (1) The plasmid genes were virtually identical to those in IncI1-ColIb plasmids from E. coli, suggesting that Salmonella and E. coli share overlapping pools of these plasmids. (2) The plasmid genes were much less polymorphic than gnd or any other known chromosomal gene from Salmonella, further suggesting horizontal transfer with rapid transmission and turnover. (3) No characteristic differences were found in either the plasmid genes or the chromosomal gene between the 1974 isolates and the Murray strains, indicating that these plasmids have been stable for at least several decades. (4) There was an excess of amino-acid replacement polymorphisms, relative to synonymous polymorphisms, in the plasmid genes, which is consistent with the hypothesis of diversifying selection among colicin-producing plasmid families. (5) The abi (abortive infection) gene present in each of the plasmids contained two single-nucleotide insertions relative to the published sequence. These result in a putative abi protein of 114 amino acids instead of 89.
Hartl, DL, and RC Lewontin. 1994. “DNA fingerprinting.” Science 266: 201; author reply 202-3.
Hartl, DL. 1994. “Forensic DNA typing dispute.” Nature 372: 398-9.
Hartl, DL, and ER Lozovskaya. 1994. “Genome evolution: between the nucleosome and the chromosome.” EXS 69: 579-92. Abstract

Intermediate between DNA sequences and broad patterns of karyotypic change there is a major gap in understanding genome structure and evolution. The gap is at the megabase level between genes and chromosomes. New methods for analyzing large DNA fragments cloned in yeast or bacterial vectors provide experimental access to genome evolution at the megabase level by enabling the assembly of megabase-size contiguous regions. Genome evolution at the megabase level can also be studied using high-resolution genetic maps. Rates and patterns of genome evolution in mammals (mouse versus humans) and Drosophila (D. virilis versus D. melanogaster) are compared and contrasted. Opportunities for research in genome evolution using the new technologies are enumerated and discussed.

Hartl, DL, DI Nurminsky, RW Jones, and ER Lozovskaya. 1994. “Genome structure and evolution in Drosophila: applications of the framework P1 map.” Proc Natl Acad Sci U S A 91: 6824-9. Abstract

Physical maps showing the relative locations of cloned DNA fragments in the genome are important resources for research in molecular genetics, genome analysis, and evolutionary biology. In addition to affording a common frame of reference for organizing diverse types of genetic data, physical maps also provide ready access to clones containing DNA sequences from any defined region of the genome. In this paper, we present a physical map of the genome of Drosophila melanogaster based on in situ hybridization with 2461 DNA fragments, averaging approximately 80 kilobase pairs each, cloned in bacteriophage P1. The map is a framework map in the sense that most putative overlaps between clones have not yet been demonstrated at the molecular level. Nevertheless, the framework map includes approximately 85% of all genes in the euchromatic genome. A continuous physical map composed of sets of overlapping P1 clones (contigs), which together span most of the euchromatic genome, is currently being assembled by screening a library of 9216 P1 clones with single-copy genetic markers as well as with the ends of the P1 clones already assigned positions in the framework map. Because most P1 clones from D. melanogaster hybridize in situ with chromosomes from related species, the framework map also makes it possible to determine the genome maps of D. pseudoobscura and other species in the subgenus Sophophora. Likewise, a P1 framework map of D. virilis affords potential access to genome organization and evolution in the subgenus Drosophila.

Capy, P, T Langin, Y Bigot, F Brunet, MJ Daboussi, G Periquet, JR David, and DL Hartl. 1994. “Horizontal transmission versus ancient origin: mariner in the witness box.” Genetica 93: 161-70. Abstract

The transposable element mariner has been found in many species of Drosophilidae, several groups of Arthropods, and more recently in Platyhelminthes as well as in a phytopathogenic fungus. In the family Drosophilidae, the distribution of mariner among species shows many gaps, and its geographical distribution among endemic species is restricted to Asia and Africa. Among mariner elements in species within and outside the Drosophilidae, the similarities in nucleotide sequence and the amino acid sequence of the putative transposase reveal many phylogenetic inconsistencies compared with the conventional phylogeny of the host species. This paper discusses the contrasting hypotheses of horizontal transfer versus ancestral origin proposed to explain these results.

Hartl, DL, and H Ochman. 1994. “Inverse polymerase chain reaction.” Methods Mol Biol 31: 187-96.
Carulli, JP, DM Chen, WS Stark, and DL Hartl. 1994. “Phylogeny and physiology of Drosophila opsins.” J Mol Evol 38: 250-62. Abstract

Phylogenetic and physiological methods were used to study the evolution of the opsin gene family in Drosophila. A phylogeny based on DNA sequences from 13 opsin genes including representatives from the two major subgenera of Drosophila shows six major, well-supported clades: The "blue opsin" clade includes all of the Rh1 and Rh2 genes and is separated into two distinct subclades of Rh1 sequences and Rh2 sequences; the ultraviolet opsin clade includes all Rh3 and Rh4 genes and bifurcates into separate Rh3 and Rh4 clades. The duplications that generated this gene family most likely took place before the evolution of the subgenera Drosophila and Sophophora and their component species groups. Numerous changes have occurred in these genes since the duplications, including the loss and/or gain of introns in the different genes and even within the Rh1 and Rh4 clades. Despite these changes, the spectral sensitivity of each of the opsins has remained remarkably fixed in a sample of four species representing two species groups in each of the two subgenera. All of the strains that were investigated had R1-6 (Rh1) spectral sensitivity curves that peaked at or near 480 nm, R7 (Rh3 and Rh4) peaks in the ultraviolet range, and ocellar (Rh2) peaks near 420 nm. Each of the four gene clades on the phylogeny exhibits very conservative patterns of amino acid replacement in domains of the protein thought to influence spectral sensitivity, reflecting strong constraints on the spectrum of light visible to Drosophila.

Hartl, DL, EN Moriyama, and SA Sawyer. 1994. “Selection intensity for codon bias.” Genetics 138: 227-34. Abstract

The patterns of nonrandom usage of synonymous codons (codon bias) in enteric bacteria were analyzed. Poisson random field (PRF) theory was used to derive the expected distribution of frequencies of nucleotides differing from the ancestral state at aligned sites in a set of DNA sequences. This distribution was applied to synonymous nucleotide polymorphisms and amino acid polymorphisms in the gnd and putP genes of Escherichia coli. For the gnd gene, the average intensity of selection against disfavored synonymous codons was estimated as approximately 7.3 x 10(-9); this value is significantly smaller than the estimated selection intensity against selectively disfavored amino acids in observed polymorphisms (2.0 x 10(-8)), but it is approximately of the same order of magnitude. The selection coefficients for optimal synonymous codons estimated from PRF theory were consistent with independent estimates based on codon usage for threonine and glycine. Across 118 genes in E. coli and Salmonella typhimurium, the distribution of estimated selection coefficients, expressed as multiples of the effective population size, has a mean and standard deviation of 0.5 +/- 0.4. No significant differences were found in the degree of codon bias between conserved positions and replacement positions, suggesting that translational misincorporation is not an important selective constraint among synonymous polymorphic codons in enteric bacteria. However, across the first 100 codons of the genes, conserved amino acids with identical codons have significantly greater codon bias than that of either synonymous or nonidentical codons, suggesting that there are unique selective constraints, perhaps including mRNA secondary structures, in this part of the coding region.

Nurminsky, DI, and DL Hartl. 1993. “Amplification of the ends of DNA fragments cloned in bacteriophage P1.” Biotechniques 15: 201-2, 206-8.
Moriyama, EN, and DL Hartl. 1993. “Codon usage bias and base composition of nuclear genes in Drosophila.” Genetics 134: 847-58. Abstract

The nuclear genes of Drosophila evolve at various rates. This variation seems to correlate with codon-usage bias. In order to elucidate the determining factors of the various evolutionary rates and codon-usage bias in the Drosophila nuclear genome, we compared patterns of codon-usage bias with base compositions of exons and introns. Our results clearly show the existence of selective constraints at the translational level for synonymous (silent) sites and, on the other hand, the neutrality or near neutrality of long stretches of nucleotide sequence within noncoding regions. These features were found for comparisons among nuclear genes in a particular species (Drosophila melanogaster, Drosophila pseudoobscura and Drosophila virilis) as well as in a particular gene (alcohol dehydrogenase) among different species in the genus Drosophila. The patterns of evolution of synonymous sites in Drosophila are more similar to those in the prokaryotes than they are to those in mammals. If a difference in the level of expression of each gene is a main reason for the difference in the degree of selective constraint, the evolution of synonymous sites of Drosophila genes would be sensitive to the level of expression among genes and would change as the level of expression becomes altered in different species. Our analysis verifies these predictions and also identifies additional selective constraints at the translational level in Drosophila.

Methods of genome analysis, including the cloning and manipulation of large fragments of DNA, have opened new strategies for uniting molecular evolutionary genetics with chromosome evolution. We have begun the development of a physical map of the genome of Drosophila virilis based on large DNA fragments cloned in bacteriophage P1. A library of 10,080 P1 clones with average insert sizes of 65.8 kb, containing approximately 3.7 copies of the haploid genome of D. virilis, has been constructed and characterized. Approximately 75% of the clones have inserts exceeding 50 kb, and approximately 25% have inserts exceeding 80 kb. A sample of 186 randomly selected clones was mapped by in situ hybridization with the salivary gland chromosomes. A method for identifying D. virilis clones containing homologs of D. melanogaster genes has also been developed using hybridization with specific probes obtained from D. melanogaster by means of the polymerase chain reaction. This method proved successful for nine of ten genes and resulted in the recovery of 14 clones. The hybridization patterns of a sample of P1 clones containing repetitive DNA were also determined. A significant fraction of these clones hybridizes to multiple euchromatic sites but not to the chromocenter, which is a pattern of hybridization that is very rare among clones derived from D. melanogaster. The materials and methods described will make it possible to carry out a direct study of molecular evolution at the level of chromosome structure and organization as well as at the level of individual genes.

Carulli, JP, DE Krane, DL Hartl, and H Ochman. 1993. “Compositional heterogeneity and patterns of molecular evolution in the Drosophila genome.” Genetics 134: 837-45. Abstract

The rates and patterns of molecular evolution in many eukaryotic organisms have been shown to be influenced by the compartmentalization of their genomes into fractions of distinct base composition and mutational properties. We have examined the Drosophila genome to explore relationships between the nucleotide content of large chromosomal segments and the base composition and rate of evolution of genes within those segments. Direct determination of the G + C contents of yeast artificial chromosome clones containing inserts of Drosophila melanogaster DNA ranging from 140-340 kb revealed significant heterogeneity in base composition. The G + C content of the large segments studied ranged from 36.9% G + C for a clone containing the hunchback locus in polytene region 85, to 50.9% G + C for a clone that includes the rosy region in polytene region 87. Unlike other organisms, however, there was no significant correlation between the base composition of large chromosomal regions and the base composition at fourfold degenerate nucleotide sites of genes encompassed within those regions. Despite the situation seen in mammals, there was also no significant association between base composition and rate of nucleotide substitution. These results suggest that nucleotide sequence evolution in Drosophila differs from that of many vertebrates and does not reflect distinct mutational biases, as a function of base composition, in different genomic regions. Significant negative correlations between codon-usage bias and rates of synonymous site divergence, however, provide strong support for an argument that selection among alternative codons may be a major contributor to variability in evolutionary rates within Drosophila genomes.

Hartl, DL, and RC Lewontin. 1993. “DNA fingerprinting report.” Science 260: 473-4.
Ayala, FJ, and DL Hartl. 1993. “Molecular drift of the bride of sevenless (boss) gene in Drosophila.” Mol Biol Evol 10: 1030-40. Abstract

DNA sequences were determined for three to five alleles of the bride-of-sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.

Ayala, FJ, BS Chang, and DL Hartl. 1993. “Molecular evolution of the Rh3 gene in Drosophila.” Genetica 92: 23-32. Abstract

Previous investigations into the evolution of the Drosophila opsin gene family are extended by inter- and intraspecific DNA sequence comparisons of the Rh3 locus in the melanogaster subgroup and D. pseudoobscura. Two separate statistical tests of the neutral-mutation hypothesis suggest that random genetic drift is responsible for virtually all of the observed amino acid replacement substitutions within the melanogaster subgroup. Analyses incorporating the D. pseudoobscura sequences are enigmatic due to the accumulation of multiple substitutions, because the McDonald-Kreitman test is not applicable to species comparisons that approach mutational saturation. However, the data from D. pseudoobscura are not inconsistent with selective neutrality. The ratio of amino acid polymorphisms within species to fixed differences between species imply that there are approximately 31 possible neutral single-step amino-acid-replacement substitutions at this locus. Synonymous substitutions are unevenly distributed among the structural domains of the Rh3 gene. Patterns of synonymous polymorphism are analyzed with respect to GC content and codon bias, and are compared to other loci from the same species.

Lawrence, JG, DL Hartl, and H Ochman. 1993. “Sequencing products of polymerase chain reaction.” Methods Enzymol 218: 26-35.
Lidholm, DA, AR Lohe, and DL Hartl. 1993. “The transposable element mariner mediates germline transformation in Drosophila melanogaster.” Genetics 134: 859-68. Abstract

A vector for germline transformation in Drosophila melanogaster was constructed using the transposable element mariner. The vector, denoted pMlwB, contains a mariner element disrupted by an insertion containing the wild-type white gene from D. melanogaster, the beta-galactosidase gene from Escherichia coli and sequences that enable plasmid replication and selection in E. coli. The white gene is controlled by the promoter of the D. melanogaster gene for heat-shock protein 70, and the beta-galactosidase gene is flanked upstream by the promoter of the transposable element P as well as that of mariner. The MlwB element was introduced into the germline of D. melanogaster by co-injection into embryos with an active mariner element, Mos1, which codes for a functional transposase and serves as a helper. Two independent germline insertions were isolated and characterized. The results show that the MlwB element inserted into the genome in a mariner-dependent manner with the termini of the inverted repeats inserted at a TA dinucleotide. Both insertions exhibit an unexpected degree of germline and somatic stability, even in the presence of an active mariner element in the genetic background. These results demonstrate that the mariner transposable element, which is small (1286 bp) and relatively homogeneous in size among different copies, is nevertheless capable of promoting the insertion of the large (13.2 kb) MlwB element. Because of the widespread phylogenetic distribution of mariner among insects, these results suggest that mariner might provide a wide host-range transformation vector for insects.

Ochman, H, FJ Ayala, and DL Hartl. 1993. “Use of polymerase chain reaction to amplify segments outside boundaries of known sequences.” Methods Enzymol 218: 309-21.