scholarly journals The relative conformational stability of the alcohol dehydrogenase alleloenzymes of the fruitfly Drosophila melanogaster

1981 ◽  
Vol 197 (1) ◽  
pp. 111-117 ◽  
Author(s):  
D R Thatcher ◽  
R Sheikh
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Conformational Stability ◽  
Effect Of Temperature ◽  
Net Charge ◽  
Subunit Dissociation ◽  
Naturally Occurring ◽  
Transverse Temperature ◽  
The Stability ◽  
Temperature Optima

The effect of temperature on four purified alleloenzymes of the alcohol dehydrogenase (Adhs, Adhf, AdhD and Adhn-5) of the fruitfly Drosophila melanogaster was investigated in detail. Initial-velocity studies showed that the naturally occurring Adhf and Adhs enzymes differed only in their temperature optima, and evidence of kinetic adaptation to high and low temperature was not apparent. All four alleloenzymes denatured irreversibly on heating purified enzyme solutions at pH 6.0. This technique revealed only small differences in thermostability between Adhf and Adhs, although the two mutant enzymes from AdhD and Adhn-5 were considerably more labile. Electrophoresis of the enzymes though a stable transverse temperature gradient proved to be a discriminating and reproducible technique. Enzymes of different net charge were compared on the same polyacrylamide gel. The Adhf enzyme was shown to be significantly less stable than the Adhs enzyme. Subunit interchange was observed at temperatures below the point at which the unfolding occurred. At pH 4.0, the Adhf/Adhs heterodimer was as stable as the Adhs homodimeric enzyme, and more stable than the Adhf homodimer. Adhn-5 and AdhD alleloenzymes were relatively thermolabile. The stability of the alleloenzymes towards urea denaturation was studied by urea-gradient electrophoresis. Only small differences in stability between the Adhf and Adhs enzymes were observed. The AdhD and Adhn-5 mutants were denatured at the same urea concentration, which was much lower than in the case of the wild-type enzymes. Except at pH 4.0, subunit dissociation could not be distinguished from the unfolding of the monomer.

scholarly journals Analysis of a Strong Suppressor of Segregation Distorter in Drosophila melanogaster

Genetics ◽  
2020 ◽  
Vol 215 (4) ◽  
pp. 1085-1105 ◽  
Author(s):  
Rayla Greenberg Temin
Keyword(s):  
Drosophila Melanogaster ◽  
Natural Populations ◽  
Drive System ◽  
Deletion Mapping ◽  
Naturally Occurring ◽  
New Information ◽  
The Stability ◽  
Insight Into ◽  
Mendelian Transmission

Segregation Distorter (SD) is a naturally occurring male meiotic drive system in Drosophila melanogaster, characterized by almost exclusive transmission of the SD chromosome owing to dysfunction of sperm receiving the SD+ homolog. Previous studies identified at least three closely linked loci on chromosome 2 required for distortion: Sd, the primary distorting gene; E(SD) (Enhancer of SD), which increases the strength of distortion; and Rsp (Responder), the apparent target of Sd. Strength of distortion is also influenced by linked upward modifiers including M(SD) (Modifier of SD) and St(SD) (Stabilizer of SD), and by various unlinked suppressors. Although Sd is known to encode a mutant RanGAP protein, none of the modifiers have been molecularly identified. This work focuses on the genetic and cytological characterization of a strong X-linked suppressor, Su(SD), capable of restoring Mendelian transmission in SD/SD+ males. Sd and its cohort of positive modifiers appear to act semiquantitatively in opposition to Su(SD) with distortion strength depending primarily on the total number of distorting elements rather than which particular elements are present. Su(SD) can also suppress male sterility observed in certain SD genotypes. To facilitate its eventual molecular identification, Su(SD) was localized by deletion mapping to polytene region 13C7-13E4. These studies highlight the polygenic nature of distortion and its dependence on a constellation of positive and negative modifiers, provide insight into the stability of Mendelian transmission in natural populations even when a drive system arises, and pave the way for molecular characterization of Su(SD) whose identity should reveal new information about the mechanism of distortion.

Aberrant splicing of a naturally occurring alcohol dehydrogenase null activity allele in Drosophila melanogaster

Genome ◽  
1990 ◽  
Vol 33 (6) ◽  
pp. 873-877
Author(s):  
Allan L. Freeth ◽  
John B. Gibson ◽  
Ann Verona Wilks
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Null Allele ◽  
Stop Codon ◽  
Aberrant Splicing ◽  
S1 Nuclease ◽  
Mature Adult ◽  
Exon 2 ◽  
Naturally Occurring ◽  
Intron 2

The DNA sequence of a naturally occurring alcohol dehydrogenase null activity allele, AdhnAC14, has eight extra nucleotides (in two groups of four) in the second intron, commencing six bases 3′ from the 5′ splice site. A stop codon was also found in exon 2. S1 nuclease protection experiments have shown that the insertions in intron 2 disrupt the correct splicing of intron 2. The null allele produces a transcript approximately 100 bases longer than the normal mature adult transcript, and the amount of the null allele transcript is only about 10% of the normal level.Key words: alcohol dehydrogenase, null allele, splicing, S1 nuclease, Drosophila melanogaster.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

Insights into the Structural Features, Conformational Stability and Functional Activity of the Olneya tesota PF2 Lectin

2020 ◽  
Vol 27 ◽  
Author(s):  
Edgar Acedo-Espinoza ◽  
Irlanda Lagarda-Diaz ◽  
Rosina Cabrera ◽  
Ana M. Guzman-Partida ◽  
Amir Maldonado-Arce ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Proteolytic Enzymes ◽  
Conformational Stability ◽  
Structural Features ◽  
Beta Sheets ◽  
Hemagglutinating Activity ◽  
Effect Of Temperature ◽  
Hydrodynamic Diameter ◽  
Bioinformatic Tools ◽  
Structural Levels

Background: The O. tesota lectin PF2 is a tetrameric protein with subunits of 33 kDa that recognizes only complex carbohydrates, resistant to proteolytic enzymes and has insecticidal activity against Phaseolus beans pest. Objective: To explore PF2 lectin features at different protein structural levels and to evaluate the effect of temperature and pH on its functionality and conformational stability. Methods: PF2 lectin was purified by affinity chromatography. Its primary structure was resolved by mass spectrometry and analyzed by bioinformatic tools, including its tertiary structure homology modeling. The effect of temperature and pH on its conformational traits and stability was addressed by dynamic light scattering, circular dichroism, and intrinsic fluorescence. The hemagglutinating activity was evaluated using a suspension of peripheral blood erythrocytes. Results: The proposed PF2 folding comprises a high content of beta sheets. At pH 7 and 25 °C, the hydrodynamic diameter (Dh) was found to be 12.3 nm which corresponds to the oligomeric native state of PF2 lectin. Dh increased under the other evaluated pH and temperature conditions, suggesting protein aggregation. At basic pH, PF2 exhibited low conformational stability. The native PF2 (pH 7) retained its full hemagglutinating activity up to 45 °C and exhibited one transition state with a melting temperature of 76.8 °C. Conclusion: PF2 showed distinctive characteristics found in legume lectins. The pH influences the functionality and conformational stability of the protein. PF2 lectin displayed a relatively narrow thermostability to the loss of secondary structure and hemagglutinating activity.

scholarly journals The Alcohol Dehydrogenase Gene Is Nested in the outspread Locus of Drosophila melanogaster

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 897-911 ◽  
Author(s):  
S McNabb ◽  
S Greig ◽  
T Davis
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Pcr Amplification ◽  
Transcription Unit ◽  
Adjacent Gene ◽  
Dehydrogenase Gene ◽  
Chromosomal Breakpoints ◽  
Splicing Patterns ◽  
Somatic Musculature

Abstract This report describes the structure and expression of the outspread (osp) gene of Drosophila melanogaster. Previous work showed that chromosomal breakpoints associated with mutations of the osp locus map to both sides of the alcohol dehydrogenase gene (Adh), suggesting that Adh and the adjacent gene Adh' are nested in osp. We extended a chromosomal walk and mapped additional osp mutations to define the maximum molecular limit of osp as 119 kb. We identified a 6-kb transcript that hybridizes to osp region DNA and is altered or absent in osp mutants. Accumulation of this RNA peaks during embryonic and pupal periods. The osp cDNAs comprise two distinct classes based on alternative splicing patterns. The 5′ end of the longest cDNA was extended by PCR amplification. When hybridized to the osp walk, the 5′ extension verifies that Adh and Adh' are nested in osp and shows that osp has a transcription unit of ≥74 kb. In situ hybridization shows that osp is expressed both maternally and zygotically. In the ovary, osp is transcribed in nurse cells and localized in the oocyte. In embryos, expression is most abundant in the developing visceral and somatic musculature.

scholarly journals INTRACISTRONIC MAPPING OF ELECTROPHORETIC SITES IN DROSOPHILA MELANOGASTER: FIDELITY OF INFORMATION TRANSFER BY GENE CONVERSION

Genetics ◽  
1974 ◽  
Vol 76 (2) ◽  
pp. 289-299
Author(s):  
Margaret McCarron ◽  
William Gelbart ◽  
Arthur Chovnick
Keyword(s):  
Drosophila Melanogaster ◽  
Information Transfer ◽  
Large Scale ◽  
Genetic Basis ◽  
Xanthine Dehydrogenase ◽  
Specific Protein ◽  
Wild Type ◽  
Electrophoretic Variation ◽  
The Stability ◽  
Recombination Experiments

ABSTRACT A convenient method is described for the intracistronic mapping of genetic sites responsible for electrophoretic variation of a specific protein in Drosophila melanogaster. A number of wild-type isoalleles of the rosy locus have been isolated which are associated with the production of electrophoretically distinguishable xanthine dehydrogenases. Large-scale recombination experiments were carried out involving null enzyme mutants induced on electrophoretically distinct wild-type isoalleles, the genetic basis for which is followed as a nonselective marker in the cross. Additionally, a large-scale recombination experiment was carried out involving null enzyme rosy mutants induced on the same wild-type isoallele. Examination of the electrophoretic character of crossover and convertant products recovered from the latter experiment revealed that all exhibited the same parental electrophoretic character. In addition to documenting the stability of the xanthine dehydrogenase electrophoretic character, this observation argues against a special mutagenesis hypothesis to explain conversions resulting from allele recombination studies.

scholarly journals Anthocyanins Recovered from Agri-Food By-Products Using Innovative Processes: Trends, Challenges, and Perspectives for Their Application in Food Systems

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2632
Author(s):  
Henrique Silvano Arruda ◽  
Eric Keven Silva ◽  
Nayara Macêdo Peixoto Araujo ◽  
Gustavo Araujo Pereira ◽  
Glaucia Maria Pastore ◽  
...  
Keyword(s):  
Food Systems ◽  
Food Industry ◽  
Biological Properties ◽  
Extraction Techniques ◽  
Naturally Occurring ◽  
Current Review ◽  
Natural Colorants ◽  
Conventional Extraction ◽  
The Stability ◽  
By Products

Anthocyanins are naturally occurring phytochemicals that have attracted growing interest from consumers and the food industry due to their multiple biological properties and technological applications. Nevertheless, conventional extraction techniques based on thermal technologies can compromise both the recovery and stability of anthocyanins, reducing their global yield and/or limiting their application in food systems. The current review provides an overview of the main innovative processes (e.g., pulsed electric field, microwave, and ultrasound) used to recover anthocyanins from agri-food waste/by-products and the mechanisms involved in anthocyanin extraction and their impacts on the stability of these compounds. Moreover, trends and perspectives of anthocyanins’ applications in food systems, such as antioxidants, natural colorants, preservatives, and active and smart packaging components, are addressed. Challenges behind anthocyanin implementation in food systems are displayed and potential solutions to overcome these drawbacks are proposed.

scholarly journals GENETIC AND BIOCHEMICAL BASIS OF ENZYME ACTIVITY VARIATION IN NATURAL POPULATIONS. I. ALCOHOL DEHYDROGENASE IN DROSOPHILA MELANOGASTER

Genetics ◽  
1978 ◽  
Vol 89 (2) ◽  
pp. 371-388
Author(s):  
John F McDonald ◽  
Francisco J Ayala
Keyword(s):  
Gene Regulation ◽  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Natural Populations ◽  
Difficult Problem ◽  
Regulatory Genes ◽  
Biochemical Basis ◽  
Evolutionary Consequence ◽  
The Third ◽  
Adh Activity

ABSTRACT Recent studies by various authors suggest that variation in gene regulation may be common in nature, and might be of great evolutionary consequence; but the ascertainment of variation in gene regulation has proven to be a difficult problem. In this study, we explore this problem by measuring alcohol dehydrogenase (ADH) activity in Drosophila melanogaster strains homozygous for various combinations of given second and third chromosomes sampled from a natural population. The structural locus (Adh) coding for ADH is on the second chromosome. The results show that: (1) there are genes, other than Adh, that affect the levels of ADH activity; (2) at least some of these "regulatory" genes are located on the third chromosome, and thus are not adjacent to the Adh locus; (3) variation exists in natural populations for such regulatory genes; (4) the effect of these regulatory genes varies as they interact with different second chromosomes; (5) third chromosomes with high-activity genes are either partially or completely dominant over chromosomes with low-activity genes; (6) the effects of the regulatory genes are pervasive throughout development; and (7) the third chromosome genes regulate the levels of ADH activity by affecting the number of ADH molecules in the flies. The results are consistent with the view that the evolution of regulatory genes may play an important role in adaptation.

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