ADH evolution and the phylogenetic footprint

The evolution of any given protein reflects the interplay between proximal selective forces involving the conservation of protein structure and function and more general populational factors that shape the action and efficiency of natural selection. In an attempt to address that interplay, we have analyzed patterns of amino acid replacement within a well-conserved molecule, alcohol dehydrogenase (ADH), in the Drosophilidae. A sliding window, moved along the protein sequence in order to quantify the extent of change at each amino acid position, reveals heterogeneous amounts of replacement across the molecule when all ADH sequences are analyzed simultaneously. Surprisingly, the replacement profile for ADH differs significantly in the melanogaster, mulleri, and Hawaiian subgroups, reflecting the imprint of the differing evolutionary histories of each of these assemblages on the evolution of this conservative molecule.Correspondence to: R.L. Dorit     

Ascidian and Amphioxus Adh Genes Correlate Functional and Molecular Features of the ADH Family Expansion During Vertebrate Evolution

The alcohol dehydrogenase (ADH) family has evolved into at least eight ADH classes during vertebrate evolution. We have characterized three prevertebrate forms of the parent enzyme of this family, including one from an urochordate (Ciona intestinalis) and two from cephalochordates (Branchiostoma floridae and Branchiostoma lanceolatum). An evolutionary analysis of the family was performed gathering data from protein and gene structures, exon–intron distribution, and functional features through chordate lines. Our data strongly support that the ADH family expansion occurred 500 million years ago, after the cephalochordate/vertebrate split, probably in the gnathostome subphylum line of the vertebrates. Evolutionary rates differ between the ancestral, ADH3 (glutathione-dependent formaldehyde dehydrogenase), and the emerging forms, including the classical alcohol dehydrogenase, ADH1, which has an evolutionary rate 3.6-fold that of the ADH3 form. Phylogenetic analysis and chromosomal mapping of the vertebrate Adh gene cluster suggest that family expansion took place by tandem duplications, probably concurrent with the extensive isoform burst observed before the fish/tetrapode split, rather than through the large-scale genome duplications also postulated in early vertebrate evolution. The absence of multifunctionality in lower chordate ADHs and the structures compared argue in favor of the acquisition of new functions in vertebrate ADH classes. Finally, comparison between B. floridae and B. lanceolatum Adhs provides the first estimate for a cephalochordate speciation, 190 million years ago, probably concomitant with the beginning of the drifting of major land masses from the Pangea. Received: 10 April 2001 / Accepted: 23 May 2001     

Induced ADH gene expression and enzyme activity in pollinated pistils of Solanum tuberosum

 The regulation of alcohol dehydrogenase (ADH) in relation to in vivo pollen tube growth of Solanum tuberosum was investigated. Adh gene expression as well as ADH enzyme activity were induced in pollinated pistils. The induced ADH isozyme in pollinated pistils is not present in pollen or anthers. The same ADH isozyme is induced in leaves submerged in water. The significance of the induction of ADH activity for pollen tube growth is discussed. Received: 13 November 1996 / Revision accepted: 8 January 1997     

ADH enzyme activity and Adh gene expression in Drosophila melanogaster lines differentially selected for increased alcohol tolerance

In Drosophila melanogaster, alcohol dehydrogenase (ADH) activity is essential for ethanol tolerance, but its role may not be restricted to alcohol metabolism alone. Here we describe ADH activity and Adh expression level upon selection for increased alcohol tolerance in different life-stages of D. melanogaster lines with two distinct Adh genotypes: Adh(FF) and Adh(SS). We demonstrate a positive within genotype response for increased alcohol tolerance. Life-stage dependent selection was observed in larvae only. A slight constitutive increase in adult ADH activity for all selection regimes and genotypes was observed, that was not paralleled by Adh expression. Larval Adh expression showed a constitutive increase, that was not reflected in ADH activity. Upon exposure to environmental ethanol, sex, selection regime life stage and genotype appear to have differential effects. Increased ADH activity accompanies increased ethanol tolerance in D. melanogaster but this increase is not paralleled by expression of the Adh gene.     

Nucleotide sequence of circular DNA molecules homologous to the 240 bp tandem repeats of the intergenic spacer of Drosophila melanogaster ribosomal DNA

We have determined the nucleotide sequence of ten 240 bp repeated sequences of the DNA intergenic spacer present in circular DNA molecules purified from D melanogaster embryos. No significant difference was found with the sequence of the chromosomal units. This suggests that most of the circular molecules homologous to the 240 bp repeats are generated by homologous recombination between adjacent chromosomal units.     

Functional Constraints of Alcohol Dehydrogenase (ADH) of Tephritidae and Relationships with Other Dipteran Species

Alcohol dehydrogenase is considered a very important enzyme in insect metabolism because it is involved (in its homodimeric form) in the catalysis of the reversible conversion of various alcohols in larval feeding sites to their corresponding aldehydes and ketones, thus contributing to detoxification and metabolic purposes. Using 14 amino acid ADH sequences recently determined in our laboratory, we constructed a three-dimensional (3D) model of olive fruit fly Bactrocera oleae ADH1 and ADH2, based on the known homologous Drosophila lebanonensis ADH structure, and the amino acid residues that have been proposed as being responsible for catalysis were located on it. Moreover, in a comparative study of the ADH sequences, the residues occupying characteristic positions in the ADH of species of the Bactrocera and Ceratitis genera (called genus-specific) as well as residues appearing only in ADH1 or ADH2 (called isozymic-specific) were defined and localized on the 3D model. All regions important for catalytic activity, such as those forming the substrate- and coenzyme-binding sites, are highly conserved in all tephritid species examined. Genus-specific amino acids are located on the outside of the protein, on loops and regions predicted to be antigenic. The higher percentage of genus-specific amino acid variation seems to be centered in the NAD adenine-binding site, located near the surface of the protein molecule. Nine of 12 isozymic-specific positions are lined along an arc on the surface of the protein, thus linking the two monomer bases of the dimer via the C-terminal interacting loops. Furthermore, the distribution of isozymic- and genus-specific amino acids on the monomer–monomer interface may have some evolutionary significance. Most amino acids predicted to be antigenic are positioned in peripheral regions of nonfunctional importance, but surprisingly, an additional antigenic region is contained within the (highly conserved in tephritids) C-terminal tail.     

人工繁殖恒河猴的血清前清蛋白、转铁蛋白、苹果酸脱氢酶和醇脱氢酶遗传多态性研究

用薄层聚丙烯酰胺凝胶电泳方法分析118头人繁殖恒河猴血清四种蛋白质和同工酶遗传基因位点的多态性,结果表明,除醇脱氢酶(ADH)为单态外,其余三个基因位点均表现多态,前清蛋白(PA)可分为AA、AB、AC、AD和BB、CC,EE七种表型,各基因的频率为A 0.85,B 0.072,C 0.042,D 0.009,E 0.034转铁蛋白(Tf)可分为CC、DD、EE、FFGG、CD、CE、CG、CH、DE、DF、DG、DH、EF、EG、EH、FG十七种表型,墓因频率为C 0.064,D 0.380,E 0.188,F 0.111,G 0.244,H 0.014,苹果酸脱氢酶(MDH)可分MDH)1-1和MDH2-1两种表型,基因频率为MDH~10.958和MDH~20.042。     

Molecular cloning and characterization of the alcohol dehydrogenase ADH1 gene of Candida utilis ATCC 9950

The alcohol dehydrogenase gene (ADH1) of Candida utilis ATCC9950 was cloned and expressed in recombinant Escherichia coli. C. utilis ADH1 was obtained by PCR amplification of C. utilis genomic DNA using two degenerate primers. Amino acid sequence analysis of C. utilis ADH1 indicated that it contained a zinc-binding consensus region and a NAD(P)⁺-binding site, and lacked a mitochondrial targeting peptide. It has a 98 and 73% identity with ADH1s of C. albicans and Saccharomyces cerevisiae, respectively. Amino acid sequence analysis and enzyme characterization with various aliphatic and branched alcohols suggested that C. utilis ADH1 might be a primary alcohol dehydrogenase existing in the cytoplasm and requiring zinc ion and NAD(P)⁺ for reaction.     

Interspecies comparison of a gene pair with partially redundant function: the rst and kirre genes in D. virilis and D. melanogaster

Abstract The D. melanogaster rst and kirre genes encode two highly related immunoglobulin-like cell adhesion molecules that function redundantly during embryonic muscle development. The two genes appear to be derived from a common ancestor by gene duplication. Gene duplications have been proposed to be of major evolutionary significance since duplicated redundant sequences can accumulate mutations without detrimental effects for the organism and leave the duplicated genes free to assume novel functions. To address the issue of conservation of the duplicated sequences and their putative redundancy, as well as to identify putative functional divergence of the paralogs during drosophilid evolution, we performed an interspecies comparison of the rst and kirre genes from D. virilis and D. melanogaster. The D. virilis genome contains orthologues of both rst and kirre and hence the duplication took place before the split of the two lineages and has subsequently been conserved. However, whilst the Rst orthologues show a high degree of sequence similarity, this similarity is lower in Kirre orthologues. Especially the intracellular domains of D. virilis and D. melanogaster Kirre sequences are highly divergent: the D. virilis kirre gene lacks the 3′-most exon present in D. melanogaster, which contains motifs conserved between kirre and rst in D. melanogaster. Hence, while each of the two genes is highly conserved at the level of its exon-intron organization, the selection forces acting on the rst and kirre coding sequences are different. These findings are discussed in the light of general evolutionary mechanisms.     

Evolution of gene transfer in bacteria

The transfer of genetic information by transformation, conjugation and transduction in bacteria occurs frequently in nature. These diverse gene transfer mechanisms in bacteria are the result of evolution and are not linked to reproduction as in eukaryotic organisms. In this review, gene transfer in bacteria will be considered from an evolutionary perspective.     

洋葱细胞质雄性不育系70及其保持系71基因组DNA的RAPD分析

采用RAPD技术,对洋葱细胞质雄性不育系70及其保持系71的基因组DNA的多态性进行分析。结果表明,在被检测的100条随机引物中,46条引物具有扩增产物,5条引物扩增出多态性条带。对这5条引物进行重复筛选和单株验证,只有G02扩增出的多态性片段,且表现稳定。推测该标记与育性有关。     

Structural and thermodynamic properties of DNA uncover different evolutionary histories

We propose an index of DNA homogeneity (IDH) based on a binary distribution model that quantifies structural and thermodynamic aggregates present in DNA primary structures. Extensive analysis of sequence databases with the IDH uncovers significant constraints on DNA sequence other than those derived from codon usage or protein function. This index clearly distinguishes between organisms of different evolutive origins and places them in disjoint domains of DNA sequence space.On sabbatical leave from Facultad de Ciencias, UNAM, MéxicoCorrespondence to: P. Miramontes     

Despite its high representation in extrachromosomal circular DNAs from Drosophila embryos,the dodecasatellite does not allow autonomous replication in cultured cells

Summary— The dodecasatellite is a 11/12 bp tandemly repeated sequence which is overrepresented, with regard to its genomic representation, in extrachromosomal circular DNAs from D melanogaster embryos. Here we show that a bacterial plasmid carrying a cluster of dodecasatellite is not able to replicate efficiently in cultured cells. This observation does not support the hypothesis that the over-representati results from an autonomous replication of dodecasatellite circular DNA molecules.     

Evolution of sea urchin non-repetitive DNA

Summary New methods have been applied to the determination of single copy DNA sequence differences between the sea urchin speciesStrongylocentrotus purpuratus, S. franciscanus, S. drobachiensis, andLytechinus pictus. The thermal stability of interspecies DNA duplexes was measured in a solvent (2.4 M tetraethylammonium chloride) that suppresses the effect of base composition on melting temperature. The lengths of duplexes were measured after digestion with S1 nuclease and correction made for the effect of length on thermal stability. The degree of base substitution that has occurred in the single copy DNA during sea urchin evolution is significantly larger than indicated by earlier measurements. We estimate that 19% of the nucleotides of the single copy DNA are different in the genomes of the two sea urchin congeners,S. purpuratus, andS. franciscanus, which apparently diverged only 15 to 20 million years ago.     

DNA2 Cooperates with the WRN and BLM RecQ Helicases to Mediate Long-range DNA End Resection in Human Cells

The 5′-3′ resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5′-3′ DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.     

Polymerase chain reaction of 2-kb cyanobacterial gene and human anti-alpha1-chymotrypsin gene from genomic DNA on the In-Check single-use microfabricated silicon chip

The microfabricated chip is a promising format for automating and miniaturizing the multiple steps of genotyping. We tested an innovative silicon biochip (In-Check Lab-on-Chip; STMicroelectronics, Agrate Brianza, Italy) designed for polymerase chain reaction (PCR) analysis of complex biological samples. The chip is mounted on a 1x3-in(2). plastic slide that provides the necessary mechanical, thermal, electrical, and fluidic connections. A temperature control system drives the chip to the desired temperatures, and a graphical user interface allows experimenters to define cycling conditions and monitor reactions in real time. During thermal cycling, we recorded a cooling rate of 3.2 degrees C/s and a heating rate of 11 degrees C/s. The temperature maintained at each thermal plateau was within 0.13 degrees C of the programmed temperature at three sensors. From 0.5 ng/microl genomic DNA, the In-Check device successfully amplified the 2060-bp cyanobacterial 16S rRNA gene and the 330-bp human anti-alpha(1)-chymotrypsin gene. The shortest PCR protocol that produced an amplicon by capillary electrophoresis comprised 30 cycles and was 22.5 min long. These thermal cycling characteristics suggest that the In-Check device will permit future development of a genotyping lab-on-a-chip device, yielding results in a short time from a limited amount of biological starting material.     

DNA-DNA dot hybridization technique used as DNA determination method in the alkaline elution analysis of DNA damage

A DNA-DNA (‘Southern’) dot hybridization technique was adapted for use as a quantitative DNA detection method during alkaline elution analysis of irradiated rat cell material. In comparison to standard microfluorometric methods, similar γ-ray-dose-response relationships were obtained with less than 1% of the cell material when the dot hybridization assay was used. When a highly repetitive, long interspersed DNA element of the rat genome is used as a hybridization probe, as few as 10⁴ cells of rat tissue or rat cell culture cells per sample with approx. 50 ng of DNA were sufficient to detect single-strand breaks and protein cross-links in the DNA of rat hepatocytes and cells of the nasal epithelium after in vitro γ-irradiation. Since highly repetative DNA elements are available from nearly all higher eukaryotes, this alternative approach of detecting DNA in alkaline elution analysis is generally proposed for tissues which yield only low amounts of cell material and/or which are difficult to label by radioactive DNA precursors.     

Evolution of programmed DNA rearrangements in a scrambled gene

Gene unscrambling in spirotrichous ciliates involves massive genome-wide DNA deletion and rearrangement events during development. During each sexual cycle, the somatic nucleus (macronucleus) regenerates from the germ line nucleus (micronucleus). Development of the polyploid somatic genome requires programmed DNA deletion of micronuclear-limited intragenic noncoding sequences and permutation and amplification of the protein-coding regions. Recent studies suggest that, despite novel insertions of endogenous transposon or foreign DNA into the germ line genome, ciliates possess a whole-genome surveillance system that guides the recapitulation of a functional somatic genome. This renders the germ line genome an extremely dynamic structure over evolutionary time. Here we describe the germ line and somatic architectures of the gene encoding alpha-telomere-binding protein in three early-diverging species (Holosticha sp., Uroleptus sp., and Paraurostyla weissei) and trace the natural history of DNA rearrangements in this gene in six species, including three previously studied oxytrichids. Comparisons of homologous coding regions between earlier and later diverging species provide evidence for fusion of scrambled germ line fragments as small as 24 bp during evolution, as well as simultaneous fragmentation and scrambling of the germ line locus and shifting of the boundaries between coding and noncoding DNA, leading to distinct gene architectures in each species. We infer an evolutionary recombination pathway that passes through identified intermediate species and gives rise to the observed patterns in all known species, capitalizing on their unique DNA rearrangement machinery and germ line flexibility.