Molecular population genetics of the<Emphasis Type="Italic">β-esterase</Emphasis> gene cluster of<Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We have investigated nucleotide polymorphism at theβ-esterase gene cluster including theEst-6 gene andψEst-6 putative pseudogene in four samples ofDrosophila melanogaster derived from natural populations of southern Africa (Zimbabwe), Europe (Spain), North America (USA: California), and South America (Venezuela). A complex haplotype structure is revealed in bothEst-6 andψEst-6. Total nucleotide diversity is twice inψEst-6 as inEst-6; diversity is higher in the African sample than in the non-African ones. Strong linkage disequilibrium occurs within theβ-esterase gene cluster in non-African samples, but not in the African one. Intragenic gene conversion events are detected withinEst-6 and, to a much greater extent, withinyEst-6; intergenic gene conversion events are rare. Tests of neutrality with recombination are significant for theβ-esterase gene cluster in the non-African samples but not significant in the African one. We suggest that the demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the pattern of nucleotide polymorphism in theb-esterase gene cluster. However there are some ’footprints’ of directional and balancing selection shaping specific distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection betweenEst-6 andψEst-6 may play an important role in the evolution of theβ-esterase gene cluster preserving the putative pseudogene from degenerative destruction and reflecting possible functional interaction between the functional gene and the putative pseudogene.Est-6 andyEst-6 may represent an indivisible intergenic complex (‘intergene’) in which each single component (Est-6 orψEst-6) cannot separately carry out the full functional role.     

Allozyme polymorphism of Mdh and α-Gpdh in Ixodes ricinus populations: comparison of borreliae-infected and uninfected ticks

Ixodes ricinus Linnaeus (Acari: Ixodidae) ticks are vectors of numerous infectious diseases in humans and animals. The allozyme variability of MDH and α-Gpdh was detected by native polyacrylamide gel electrophoresis in I. ricinus natural populations in three localities in Serbia. Four alleles of Mdh locus (MDH 1, MDH 2, MDH 3 and MDH X) and four alleles of α-Gpdh locus (VS, S, F and VF) were detected. Interpopulation differences in Mdh and α-Gpdh allele frequencies were statistically insignificant. Significant difference in α-Gpdh allele frequencies between males and females was recorded in the largest sample only. Differences in allele frequencies, detected between borreliae-infected and uninfected I. ricinus ticks, were close to the level of statistical significance, especially for α-Gpdh locus. Clear significant difference appeared in females when sexes were tested separatelly (P = 0.037). It is interesting that genotypes containing rarer alleles (MDH 1 and S) were infected in higher proportion in comparison to other genotypes. Our results point towards a possible role of Mdh and α-Gpdh loci in I. ricinus ticks in the determination of energy requirements for host seeking. Sex differences in α-Gpdh allele frequencies suggest that selective pressure, concerning efficiency of reserve materials utilisation, points to α-Gpdh rather than to Mdh locus.     

A MOLECULAR APPROACH TO THE ROLE OF HISTORICITY IN EVOLUTION. I. EXPERIMENTAL DESIGN WITH ENZYME POLYMORPHISM IN DROSOPHILA MELANOGASTER

I investigate the role of the past history (characterized by two inverse sequences of three environments) experienced by Drosophila melanogaster populations, on the allozyme frequencies at four loci (α-Gpdh, Adh, Est-6, Pgm) in a common and constant final environment. The only locus for which an effect of past history was detected is Adh. Although not unambiguous, this result is discussed in terms of interactions between this locus and other polymorphic loci. For Est-6 and Pgm, no influence of past history was seen. As for α-Gpdh, the substantial heterogeneity between replicate populations belonging to the same historical series is probably due to a hitch-hiking effect of genes surrounding this locus. More generally, the role of historicity (i.e., the factors originating from the past history of a population and being accountable for its contemporary evolution) in creating genetic diversity between populations and among species is discussed.     

Variation in alcohol dehydrogenase activity in vitro in flies from natural populations ofDrosophila melanogaster

Alcohol dehydrogenase (ADH) activity variation in male flies taken directly from seven natural populations ofDrosophila melanogaster is largely accounted for by segregation of alleles at theAdh structural gene locus. There was little overlap in the ADH activities ofAdh ^F andAdh ^s homozygotes. Body weights varied only slightly betweenAdh genotypes and contributed little to ADH variation. Between and within population variation in ADH activity and ADH protein in flies in the wild is mainly due to the relative frequencies ofAdh ^F andAdh ^s.     

Analysis of microdifferentiation in a Spanish cellar population of Drosophila melanogaster

Variation in Adh and Gpdh-1 gene frequencies has been used to check for microdifferentiation in Spanish samples of Drosophila melanogaster inside and outside a wine cellar. Flies were collected after vintage and after overwintering respectively; within each period samples were taken on up to five consecutive days each month. Variation of gene frequencies of Adh and Gpdh-1 can be considered random when samples collected each month are taken into account. When mean monthly frequencies are considered, Gpdh-1 does not show any significant variation all over the year; yet, variation of the frequency of Adh ^S shows a cyclical pattern, its frequency being maximum at the end of the summer and minimum after overwintering. Due to the parallel change of the frequency of the inversion In(2L)t and the Adh ^S allele, no decision can be made whether the Adh locus itself or the inversion are responsible for the changes.     

Perturbation of gene frequencies in a natural population of Drosophila melanogaster: evidence for selection at the Adh locus

The cellar population of Drosophila melanogaster at the Chateau Tahbilk Winery (Victoria, Australia) was perturbed for alcohol dehydrogenase (Adh) gene frequencies. Phenol oxidase (Phox) frequencies were also perturbed and monitored as a control. Subsequent gene frequency changes, together with information on population structure, indicated that selection acted on the chromosome regions of both loci. Adh gene frequencies returned to preperturbation levels in a predictable manner. A model in which the relative fitness of Adh phenotypes was determined by temperature-dependent specific activities of enzymes of Adh genotypes adequately accounts for the rate of gene frequency change at this locus. Thus temperature behaves as a selective agent in modulating Adh gene frequencies in this cellar environment.     

The fate of several migrant genes in isolated populations of Drosophila melanogaster

Sepia-eyed flies carrying the slow electrophoretic variant of either Est-6 or Adh were introduced in low numbers and at infrequent intervals into populations of wildtype flies (+ ^se /+ ^se ) that were also homozygous for the fast moving variant of either Est-6 (50 populations) or Adh (50 populations). After 24 generations, the frequency of the sepia alleles was approximately 25%, although there was considerable variation from population to population. The fate of the Est-6 slow allele corresponded closely to that of sepia (which is located ten map units distant), although one population retained the slow allozyme variant but rejected sepia. The Adh slow allele was also retained by many populations. A number of them retained Adh-S but not sepia, and vice versa; these loci are on different chromosomes. The advantage of sepia heterozygotes was estimated to be about twice that of wildtype homozygotes. The data suggest that the selective advantage resides not with the sepia locus itself, but with a nearby chromosomal region.Financial support for work reported here was supplied under grant number GM24850, National Institutes of Health.     

A biochemical genetic study of alcohol dehydrogenase isozymes of the medfly,Ceratitis capitata wied

A concerted effort is under way to analyze, at the genetic, biochemical, and molecular level, theAdh gene system in the medflyCeratitis capitata, an important agricultural pest. The isoelectric focusing (IEF) pattern of alcohol dehydrogenase (ADH) of the medfly demonstrates the presence of two well-differentiated, genetically independent dimeric proteins, called ADH-1 and ADH-2. These proteins do not exhibit interlocus heterodimeric isozymes, and the genes are not controlled coordinately during development,Adh ₁ andAdh ₂ being expressed mainly in muscle or in fat body and ovary, respectively. From the intensity of the IEF isozyme patterns, primary alcohols are judged to be better substrates than secondary alcohols, in contrast withDrosophila melanogaster ADH, and ethanol is probably the most efficient substrate for both sets of isozymes. The isoelectric points of ADH-1 (pI=5.4) and ADH-2 (pI=8.6) are different fromD. melanogaster ADH (pI=7.6), but the medfly ADH-1 has a native molecular weight (approx. 58 kD) close to that ofD. melanogaster. A population survey of samples both from laboratory strains and from wild geographically different populations showed that theAdh ₁ locus is more polymorphic thanAdh ₂. The most variable populations are from Africa, the supposed source area of the species. Further, a case of selection at theAdh ₁ locus under laboratory conditions is reported. The hypothesis ofAdh gene duplication and the degree of similarity between medfly andDrosophila ADH are also discussed. This research was supported mainly by National Research Council of Italy, Special Project RAISA, Sub-project No. 2, Paper No. 342. Grants from the International Atomic Energy Agency, Vienna, Austria, from European Communities Commission, Second R & D Programme, “Science and Technology for Development,” and from the Italian Ministry of University and Scientific Research and Technology (“Funds 40%”) also supported this work. This paper was written when the senior author was on leave of absence at the IMBB, Crete, Greece; he was financially supported by an ECC Senior Fellowship.     

Effect of aPMR1 disruption on the processing of heterologous glycoproteins secreted in the yeastSaccharomyces cerevisiae

TheSaccharomyces cerevisiae PMR1 gene encodes a Ca²⁺-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α₁-antitrypsin (α₁-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α₁-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.     

Genetics of a tissue esterase polymorphism (Est-6) in the rabbit (Oryctolagus cuniculus)

Genetic analysis of a polymorphic tissue esterase revealed a new locus (Est-6) with two alleles (Est-6 ^a andEst-6 ^b) on linkage group VI of the rabbit.Est-6 is closely linked to theEst-1,2,4 cluster. Esterase ofEst-6 is found in many organs, particularly in liver and small intestine, but not in erythrocytes and serum.Est-6 esterase hydrolyzes -naphthyl acetate and butyrate, naphthol AS-D acetate, indoxyl acetate, and butyrate as well as 5-bromoindoxyl acetate,N-acetyl-l-alanine--naphthyl ester but not 4-methylumbelliferyl acetate and fluorescein diacetate. The enzyme is inhibited by bis-p-nitrophenyl phosphate and eserine but not byp-chloromercuribenzoate. It was classified as a carboxylesterase (EC 3.1.1.1). Based on chromosomal localization, tissue distribution, substrate specificity, inhibitor sensitivity, and range ofpI's, rabbitEst-6 is assumed to be homologous with mouseEs-7.The contribution of Dr. O. von Deimling (No. 59) was supported by the Deutsche Forschungsgemeinschaft (De 315/2-2).     

Involvement of two differenturf-s related mitochondrial sequences in the molecular evolution of the CMS-specificS-Pcf locus in petunia

In petunia, a mitochondrial (mt) locus,S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). TheS-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF,Pcf, contains parts of theatp9 andcoxII genes and an unidentified reading frame,urf-s. The second and third ORFs contain NADH dehydrogenase subunit 3 (nad3) and ribosomal protein S12 (rps12) sequences, respectively. Thenad3 andrps12 sequences included in theS-Pcf locus are identical to the corresponding sequences on the mt genome of fertile petunia. In both CMS and fertile petunia, only a single copy ofnad3 andrps12 has been detected on the physical map of the main mt genome. The origin of theurf-s sequence and the molecular events leading to the formation of the chimericS-Pcf locus are not known. This paper presents evidence indicating that two different mt sequences, related tourf-s and found in fertile petunia lines (orf-h and Rf-1), might have been involved in the molecular evolution of theS-Pcf locus. Southern analysis of mtDNA derived from both fertile and sterile petunia plants suggests that one of theseurf-s related sequences (showing 100% homology tourf-s and termedorf-h) is located on a sublimon. An additional, low-homologyurf-s related sequence (Rf-1) is shown to be located on the main mt genome 5′ to thenad3 gene. It is, thus, suggested that the sequence of events leading to the generation of theS-Pcf locus might have involved introduction of theorf-h sequence, via homologous recombination, into the main mt genome 5′ tonad3 at the region where the Rf-1 sequence is located. Contribution [No. 1581-E (1995 series)] from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel 50 250     

Heterosis in the flour moth,Ephestia kühniella

Four lines of Ephestia kühniella each homozygous for one of three different allozyme alleles at the Est-2 locus and two alleles at the Adh locus were crossed in order to study the extent of somatic, reproductive and adaptive heterosis in F₁ hybrids in comparison with the mean performance of the simultaneously reared inbred parent lines. With regard to adult weight and wing length (somatic heterosis) hybrids exhibit maximally 20% (males 10%) and 9% heterosis, respectively. As concerns the production of eggs and hatched larvae (reproductive heterosis) hybrids exceed the parental mean by 90% and 200%. Adaptive heterosis is realized by a shorter development period of the hybrids (maximally by 30%) as well as by significant lower variance of all metrical characters studied. In the F₂ the degree of heterosis diminishes. There is neither an excess of heterozygotes among the segregating allozyme genotypes nor superior performance of the heterozygotes concerning any one of the traits studied. Therefore, it is concluded that the pronounced heterosis in F₁ is not a single-gene-heterosis operating at the Est-2 and the Adh-locus.     

Polymorphism of T-cell receptor genes among laboratory and wild mice: Diverse origins of laboratory mice

Southern blots of genomic DNA from 23 strains of laboratory mice and 19 individual wild mice were examined for restriction fragment length polymorphisms in their loci encoding the T-cell receptors (Tcr): the constant regions of the α, β, and γ chains (C _α,C _β, andC _γ) and a variable region family of the β chain (V _β8). Only a few polymorphisms were observed for each locus in the laboratory mice after using three restriction enzymes,Bam HI,Eco RI, andHind III. All the laboratory mice examined fall into one of two types for theC _α,C _β andV _β8 loci and one of three types for theC _γ. These types are found in some of the wild mice studied, indicating that they were already present in the founder mice of laboratory mouse strains. In contrast, theTcr genes are highly polymorphic among wild mice. Analysis of the polymorphisms in these loci suggests that laboratory mice have inherited their genes not only fromMus musculus domesticus, but also from other subspecies, and much more than previously believed from Asian subspecies.     

Evolution du polymorphisme enzymatique dans des populations expérimentales de Drosophila melanogaster III. Déséquilibre de linkage entre les locus Adh et α-Gpdh

Evolution of enzyme polymorphism in experimental populations of Drosophila melanogaster III. Linkage disequilibrium between alleles of the Adh and -Gpdh loci — The evolution of gametic frequencies and linkage disequilibria between alleles of the Adh and -Gpdh-loci was followed in two experimental populations of Drosophila melanogaster maintained at different temperatures. The results observed were compared with those expected on the basis of theoretical models of gametic selection. Gametic fitness values were estimated from the analysis of the productivity of the different homozygous genotypes.Our experimental results indicate that the selection favours an association between the alleles Adh-F and -Gpdh-S, but it is impossible to generate and/or maintain a stable linkage disequilibrium between the two alleles.     

The “Sardinian”HLA-A30,B18,DR3,DQw2 haplotype constantly lacks the21-OHA andC4B genes. Is it an ancestral haplotype without duplication?

TheC4 and21-OH loci of the class III HLA have been studied by specific DNA probes and the restriction enzymeTaq I in 24 unrelated Sardinian individuals selected from completely HLA-typed families. All 24 individuals had theHLA extended haplotypeA30,Cw5,B18, BfF1,DR3,DRw52,DQw2, named “Sardinian” in the present paper because of its frquency of 15% in the Sardinian population. Eighteen of these were homozygous for the entire haplotype, and six were heterozygous at theA locus and blank (or homozygous) at all the other loci. In all completely homozygous cells and in four heterozygous cells at theA locus, the restriction fragments of the21-OHA (3.2 kb) andC4B (5.8 kb or 5.4 kb) genes were absent, and the fragments of theC4A (7.0 kb) and21-OHB (3.7 kb) genes were present. It is suggested that the “Sardinian” haplotype is an ancestral haplotype without duplication of theC4 and21-OH genes, practically always identical in its structure, also in unrelated individuals. The diversity of this haplotype in the class III region (about 30 kb less) may be at least partially responsible for its misalignment with most haplotypes, which have duplicatedC4 and21-OH genes, and therefore also for its decreased probability to recombine. This can help explain its high stability and frequency in the Sardinian population. The same conclusion can be suggested for the Caucasian extended haplotypeA1,B8,DR3 that always seems to lack theC4A and21-OHA genes.     

Hemoglobin polymorphism in macaques with reference to the evolution ofMacaca fascicularis andMacaca mulatta

Blood samples were collected fromMacaca fascicularis andMacaca mulatta living in indoor breeding groups and investigated electrophoretically. Hemoglobin polymorphism was observed in both species. Isoelectric focusing was performed on urea denaturated samples to test the hypothesis of a site duplication at theα-chain locus inM. fascicularis (Barnicot et al., 1970). The results of our investigations do not support the above mentioned hypothesis. Only one locus coding theα-chain was detected, and this is under the control of two alleles. Evolutionary events at the molecular level are discussed, as well asWheatley's hypothesis (1980) that malaria was an important force behind divergence in both species. InM. fascicularis hemoglobin variants might be similarily connected with malaria resistance as in man. We suggest that this was not an important process behind speciation in macaques.     

TheAdh genomic region ofDrosophila ambigua: Evolutionary trends in different species

Summary The study of individual genes is essential to a comprehensive understanding of genome evolution. The wealth of information on alcohol dehydrogenase (Adh) inDrosophila makes this gene particularly suitable for such analysis. We have characterized more than 4 kb of the genomicAdh region inDrosophila ambigua and compared this region toDrosophila mauritiana andDrosophila pseudoobscura. The presence of two genes,Adh and 3ORF (open reading frame), has been confirmed and some of their essential features have been inferred from primary structural analysis. Inter- and intraspecific comparisons have led us to support that both genes may have diverged from an ancient precursor. They appear to be evolving independently, and show a species-specific pattern. TheAdh in theobscura group species lacks amino acids three and four when compared to the species of themelanogaster group and has accumulated most of its amino acid replacements in the third exon. Neither characteristic is observed when any other group species are compared, which suggests that these may be particular features of the evolution of theobscura group. The 3ORF is highly conserved among the three species analyzed, although variability in the length of the third exon and the nucleotide substitution rate, which is much higher than inAdh, are worth noting. According to our data, both mutation/fixation rates and the distribution of mutations vary over time, which makes it difficult to predict the evolutionary dynamics of specific genome regions.     

The biallelica mating type locus ofUstilago maydis: remnants of an additional pheromone gene indicate evolution from a multiallelic ancestor

Thea mating type locus ofUstilago maydis contains the structural genes for a pheromone-based cell recognition system that governs fusion of haploid cells. The locus exists in two alleles, termeda1 anda2. We have completed the analysis of the nucleotide sequences unique toa1 anda2. Within these dissimilar regions we find two short patches of DNA sequence similarity. Interestingly, one of these segments corresponds to the transcribed region of thea1 pheromone precursor. As a result of multiple nucleotide exchanges this sequence does not code for a functional product. The existence of a second pheromone gene in thea2 allele suggests that the present locus had a multiallelic ancestor. In addition, we describe the presence of two additional genes in thea2 allele. We have investigated the role of these genes during mating and pathogenic development and speculate that they might affect mitochondrial inheritance.     

hsp 83 mutation is a dominant enhancer of lethality associated with absence of the non-protein codinghsrω locus inDrosophila melanogaster

Thehrsω or the 93D heat shock locus ofDrosophila melanogaster, which does not code for any protein, has an important role in development since nullosomy of this locus in transheterozygotes for two overlapping deficiencies, viz.,Df(3R) e ^Gp4 (eGp4) andDf(3R)GC14 (GC14), is known to cause a high (∼ 80%) mortality with the small number of escapee nullosomic flies being sterile, weak and surviving for only a few days. We now show that a majority of thehsrω-nulosomics die as embryo and that the 20% escapee embryos develop slower compared to their sibs carrying either one or two copies of thehsrω locus but after hatching survive to pupal/imago stage. Most interestingly, we further show that when onehsp83 mutant allele (hsp83 ^e4A) is introduced ineGp4/GC14 trans-heterozygotes, practically none of thehsrω-nullosomic embryos develop beyond the 1st instar larval stage. The specificity of this interaction betweenhsp83 andhsrω genes was further confirmed by examining the effect of thehsp83 mutant allele on other mutations in the 93D cytogenetic region. Therefore, we conclude that thehsp83 mutation acts as a dominant enhancer of the lethality associated with nullosomy for thehsrω gene. The observed genetic interaction between these two members of the heat shock gene family during normal embryonic development ofDrosophila reveals novel aspects of their biological functions.     

Therps16 intron and the phylogeny of theRubioideae (Rubiaceae)

The phylogeny of the subfamilyRubioideae (Rubiaceae) was estimated from sequence variation in therps16 intron (cpDNA) in 143 ingroup and 5 outgroup taxa. The analysis largely confirms a recent one based onrbcL sequences, but branch support is often much stronger. Three of the traditional subfamilies are supported,Rubioideae, Cinchonoideae s. str., andIxoroideae s. l. while there is no support forAntirheoideae. TheRubioideae are the sister group of all otherRubiaceae and comprise the tribesAnthospermeae, Coccocypseleae, Cruckshanksieae, Coussareeae, Gaertnereae, Hedyotideae, Knoxieae, Morindeae, Ophiorrhizeae, Paederieae, Pauridiantheae, Perameae, Psychotrieae, Rubieae, Spermacoceae, Theligoneae, andUrophylleae. TheHamelieae andHillieae belong to theCinchonoideae. Rachicallis andSiemensia should be transferred from theHedyotideae to theCinchonoideae. ThePauridiantheae, Urophylleae, Ophiorrhizeae, andRaritebe form the basalmost subclade of theRubioideae. The second basalmost clade consists of the generaLasianthus andPerama. The third basalmost clade consists of the tribesCoussareeae, Coccocypseleae andCruckshanksieae, and the generaDeclieuxia andHindsia. The tribesKnoxieae, Anthospermeae, Argostemmateae, Paederieae, Theligoneae, Rubieae, Hedyotideae, andSpermacoceae are members of one clade. TheKnoxieae are monophyletic ifOtiophora, Otomeria, andPentas are included. The tribeAnthospermeae is supported as monophyletic, but its subtribes are not. ThePaederieae, together withTheligonum, form a paraphyletic grade basal to theRubieae. TheHedyotideae, includingSchismatoclada, form a grade at the base of theSpermacoceae. TheGaertnereae are monophyletic and distinct from thePsychotrieae. TheMorindeae are monophyletic and includeDamnacanthus andMitchella. Schradera is the sister group of theMorindeae. ThePsychotrieae are monophyletic when theGaertnereae, Lasianthus, andDeclieuxia are excluded. The recognition of a subtribeHydnophytineae leaves the rest of thePsychotrieae paraphyletic.Psychotria is paraphyletic with respect to all other genera of the tribe. Approximately 50 genera are here classified for the first time based on molecular data.