AMINO ACID AND PEPTIDE METABOLISM AS INFLUENCED BY GROSS .
This dissertation has beenmicrofilmed exactly as received69-5411ELLIS, Albert Temple, 1930AMINO ACID AND PEPTIDE METABOLISM ASINFLUENCED BY GROSS GENE REARRANGEMENTSIN DROSOPHILA PSEUDOOBSCURA.University of Arizona, Ph.D., 1968Biology-GeneticsUniversity Microfilms, Inc., Ann Arbor, Michigan
/AMINO ACID AND PEPTIDE METABOLISM AS INFLUENCEDBY GROSS GENE REARRANGEMENTS IN DROSOPHILAPSEUDOOBSCTJHAby.le pLfe.Albert TV EllisA Dissertation Submitted to the Faculty of theDEPARTMENT OP BIOLOGICAL SCIENCESIn Partial Pulfillment of the RequirementsFor the Degree ofDOCTOR OP PHILOSOPHYIn the Graduate CollegeTHE UNIVERSITY OP ARIZONA1968
THE UNIVERSITY OF ARIZONAGRADUATE COLLEGEI hereby recommend that this dissertation prepared under mydirection byentitledAmlno.Albert? T»EllisAcid and Peptide Metabolism as Influenced by GrossGene Rearrangements In Drosophlla pseudoobsoura.be accepted as fulfilling the dissertation requirement of thedegree ofDoctor of Philosophyz .,Dissertation 6irectorDate)%y/After inspection of the final copy of the dissertation, thefollowing members of the Final Examination Committee concur inits approval and recommend its acceptance:*12fi4s.3L . if 7oJ / 9a v"sxr tw na7"/?7J)(y('V2%-&1.IThis approval and acceptance is contingent on the candidate sadequate performance and defense of this dissertation at thefinal oral examination. The inclusion of this sheet bound intothe library copy of the dissertation is evidence of satisfactoryperformance at the final examination.
STATEMENT BY AUTHORThis dissertation has been submitted in partialfulfillment of requirements- for an advanced degree at TheUniversity of Arizona and is* deposited in the UniversityLibrary to be made available to borrowers under rules ofthe Library.Brief quotations from this dissertation are allow able without special permission, provided that accurateacknowledgment of source is made. Requests for permissionfor extended quotation from or reproduction of this manu script in whole or in part may be granted by the head ofthe major department or the Dean of the Graduate Collegewhen in his judgment the proposed use of the material isin the interests of scholarship. In all other instances,however, permission must be obtained from the author.SIGNED:7
ACKNOWLEDGEMENTSThe author wishes to express his appreciation toDr. Theodosius Dobzhansky for supplying the threeDrosophila stocks used in this study and to Drs. KaoruMatsuda and Stanley Alcorn for their assistance with manyof the experimental techniques.The author also wishes to extend his thanks toDr. Robert M. Harris for his constant help, guidance andencouragement throughout the performance of these experi ments.iii
TABLE OF CONTENTSPage1.Liat of Tablesvl2.List of Illustrationsvii3.Abstractviii!(.Introduction .15*Materials and Methods8Experimental PopulationsEstablishment and Screening ofPopulations8Growth Conditions9Amino Acid Analysis10Extraction Procedures .10Chromatographic Techniques11Protein Differences6.812Extraction13Electrophoresis11 Results16Amino Acids16Quantitative Differences16Qualitative Differences18Amino Acids Found in allMetamorphic Stages ofSpecific Genotypesiv18
Pag Amino Acids Found only inMetamorphio Stages ofInversion Iiomozygotes19Amino Acids Found only inMetamorphio Stages ofInversion Heterozygotes .20Protein Analysis22Inversion Homozygotes23Inversion Heterozygotes21 .7.Discussion8.Appendix AI4.89.Appendix B71Literature Cited8 10.
;; ./- : »LIST OP TABLESTable No.1.PageNumber of individuals of each metamorphiostage of JD. pseudoobBcura used in aminoaoid extractions .26Number of dropB of amino acid extracts spottedon thin layer chromatographic plates273.Amino acid standards28i .Minimum concentrations.in Ag of standard aminoacids detectable with ninhydrin using a onedimensional technique on Silica Gel 0 .29Yield in mg of lyophilyzed protein extractof inversion homozygotes and inversionheterozygotes of D. pseudoobscura30Number of different amino acids present in thevarious metamorphic stages of inversionhomozygotes raised at 16 C and 25 C .31Number of different amino acids present in thevarious metamorphic stages of inversionheterozygotes raised at 16 C and 25 C32Amino acids present in the various metamorphicstages of inversion homozygotes and inversionheterozygotes raised at lt " G and 2 C332.6.7.8.Appendix TablesTables 1-10. Inversion Homozygotes 1Tables 11-20. Inversion Heterozygotes .61vi
«LIST OP ILLUSTRATIONSFigure1.2.3.1 .PageProtein fractions from T/ST# CH/CH and AR/ARinversion homozygotes raised at 16 C 3U-Protein fractions from ST/ST, CH/CH and AR/ARinversion homozygotes raised at 2 P C. .3S Protein fractions from ST/CH, ST/AR and CH/ARinversion heterozygotes raised at 16 C.36Protein fractions from ST/CH, ST/AR and CH/ARinversion heterozygotes raised at 25 C. .37Appendix Illustrations72Illustrations 1-6. Protein fractions ofinversion homozygotes73Illustrations 7-12. Protein fractionsinversion heterozygotes79vii
ABSTRACTAMINO ACID AND PEPTIDE METABOLISM AS INFLUENCED BY GROSSGENS REARRANGEMENTS IN DROSOPHILA PSEUDOOBSCURAbyAlbert T. ElliaAmino acid and peptide metabolism in isogenicstrains of Standard, Arrowhead and Chiricahua inversionraces of Drosophila pseudoobscura have been studied bymeans of thin layer chromatography and electrophoresis.Both qualitative and quantitative differences among thezygote, 72 hour old larvae, 21 hour old pupae, 72 hour oldpupae and 21 hour old imagoes of the ST/ST, AR/AR and CH/CHinversion homozygotes have been detected at both 16 C and25 C.Quantitatively those metamorphic stages raised at16 C generally contained more extractable free amino acidsthan those individuals raised at 25 C.In the ST/ST and CH/CH inversion homozygotes raisedat both 16 C and 25 C the number of extractable aminoacids increases from the zygote stage through the 2l . hourpupal stage followed by a decrease in the number of aminoacids through the 72 hour pupal stage and then an increasein the number of amino acids In the 24 hour imago stage.viii
ixIn the AR/AR inversion homozygote this general pattern wasslightly altered,A deorease in the number of extractableamino acids oocurred between the ?2 hour pupal and 2ij. hourimago stage.The quantitative amino acid fluctuationsoccurring among the various met amorphic stages of theinversion homozygotes were not generally found among thesame metamorphic stages of the inversion heterozygotesrraised at both 16 C and 25 C. A quantitative patternsimilar to that found in the inversion homozygotes wasfound only in the ST/CH inversion heterozygote raised at2 C.Numerous qualitative amino acid differences werealso found.Of the twenty amino acids commonly occurringin proteins only alanine and tyrosine were not detected inany metamorphic stage of any genotype at either 16 G or25 G«Th remaining eighteen commonly occurring aminoacids oocurred in three general categories.Four aminoacids were found in all metamorphic stages of certaingenotypes of both inversion homozygotes and inversionheterozygotes.Many amino acids were restricted tometamorphic stages in either inversion homozygotes orinversion heterozygotes, however, no pattern as to genotype,metamorphic stage or temperature was evident.Thirty distinctly resolved proteins were consistentlyobserved in a 50 ammonium sulfate fraction obtained from
X.24 hour old inversion homozygot imagoes raised at both16 C and 25 C.Eleven proteins were observed in the ST/STgenotype, eleven in the CH/CH genotype and eight in the AR/ARgenotype.Two proteins were distinctly common to all threeinversion homozygotes, one additional protein oocurred inboth the CH/CH and AR/AR genotypes while yet another proteinwas found in both the 5T/3T and AR/AR inversion horaoaygotesat both 16 C and 25 C.Thirteen of the thirty proteinswere found occurring only once among the three inversionhomozygotes.Thirty-seven distinct proteins were consistentlyobserved in the inversion heterozygotes raised at both 16 Cand 2 C.Fourteen proteins were found in the ST/GH hybrid,twelve in the ST/AR hybrid and eleven in the CH/AR hybrid.A number of proteins common to the various hybrids wereobserved.Evidence is presented which indicates that manyof these proteins may represent hybridization of electrophoretic variants.
INTRODUCTIONThe genetic control of physiological processes hasbecome well established in biological literature during thepast twenty-six years.The elegant work of Beadle (19i(.6)»Beadle and Tatum (19 .1), Horowitz (195 0) and a host of otherinvestigators with Neurospora crassa established the role ofthe gene in the enzymatic control of the individual stepsin biochemical reactions.The structure and specificity ofeach polypeptide or enzyme is determined by a different gene(Horowitz, 19ii8).Studios by Ingram (1956) and Hunt andIngram (I960) on human hemoglobin and recent studies ontryptophan synthetase from Escherichia coli by Yanofsky,Helinski and Maling (1961), Henning and Yanofsky (1963) andYanofsky ert al (196fy.) established the role of the gene indetermining the primary structure of proteins.Studies byJacob and Monod (1961) have further strengthened the geneprotein relationship by the discovery of genetic mechanismsfor the regulation of protein synthesis.Hubby (1963) and Hubby and Throckmorton (1965) haveutilized genetically controlled protein structure to studythe evolutionary relationships in the virilis group ofDrosophila and protein differences in Etrosophilamelanogaster.1
The most thorough understanding of polymorphism insexual species has been obtained through studies of theinverted sections of chromosomes in various species ofDrosophila.The first inversions were detected' in Drosophilamelanogaster through supression of crossover gametes ininversion heterozygotes (Sturtevant, 1926, 1931)-Th study of inversions was facilitated by the technique ofobserving the giant chromosomes in the salivary glands ofthe larval stage (Painter, 193U)«The objective of the present investigation involvinginversion races of Drosophila pseudoobscura is to study theeffect of large gene rearrangements on amino acid andpeptide metabolism.The three principle inversion racesutilized in this study offer an opportunity to analyze theeffects of gross chromosome changes on the physiology of anorganism.No studies of this nature appear in the literaturealthough Dobzhansky and other investigators have describedthe effects of these inversions on the adaptability of theseinversion races to various environmental conditions.Dobzhansky and Sturtevant (193 ) and Dobzhansky andEpling (191 1.) have shown that most natural populations of*two closely related species, Drosophila p3eudoob3cura andDrosophila persimills are mixtures of individuals withdifferent gene arrangements in their third chromosomes.
3Sixteen different gene arrangements are known in Drosophilap3eud.Q0b3cu.ra and eleven are known in Rrosophila persimilis.Only one arrangement termed standard is common to bothspecies.All of these arrangements are interrelated asoverlapping inversions.Although a complete collectionof these inversions is not found in any natural population,*in some localities up to eight inversions occur togetherand both inversionhomozygotes and inversion heterozygoteaare found, giving rise to polymorphism.Chromosomal poly-»morphism due to inversions has been found in naturalpopulations of about thirty 3pecies of Drosophila.Of thespecies examined in detail only D. virilis and D. hydeido not possess chromosomal polymorphism (Warters, 191 14-).Studies with experimental populations maintained inpopulation cages have shown that a stable equilibrium willbe established under the experimental conditions with theinversion races present in the gene pool in definite frequen cies.The establishment of stable equilibria in experimentalpopulations is the rule whenever the competing inversionraces are from the same geographical location, (Dobzhansky,1914.7a, 19i4.7b; Dobzhansky and Wallace, I9I4-.8)-A similarsituation has been found by Spei3s (1950) in D. persimilisand by Levitan (1951) in D. robusta.The adaptive values ofthe inversion races are extremely sensitive to environmentalconditions.Population experiments containing Standard and
Chiricahua inversionraces maintained at 25 G exhibit achange in inversion frequencies and establishment of astable equilibrium regardless of the initial frequenciesof the inversion types.If these inversion types are raisedat 16 C, however, no change in the relative frequencies isobserved and the adaptive values of the homozygote*s andheterozygotes are similar (Wright and Dobzhansky, 19 6).Cyclic seasonal changes in the frequencies of certaininversion types takeplace in natural populations of somelocalities of Mt. San Jacinto in California (Dobzhansky,19i 3)*Dubinin and Tiniakov (1945# 19it-6b and 1946c) haveobserved cyclic seasonal changes in the frequencies ofchromosome types in D. funebris.Small seasonal changesin the frequency of chromosomal inversions have also beenfound in populations of D. robusta (Carson and Stalker,1949).The inverted sections of the third chromosome ofP.' pseudoobscura may produce their adaptive advantage bymodifying the physiology of the carriers either by positioneffect or by possessing mutant genes which differ -fromthose in the chromosomes with the alternate inversion types.The effect may also be produced by a combination of both.Dobzhansky (1950c) has been able to show by means ofexperimental populations that when inversions from differentgeographical locations are made to compete with one another
*the inversion heterozygote shows an adaptive advantage inonly a very few cases.The adaptive value of the heter ozygote is, therefore, not determined by the inversionalone but at least in part by the genes located withinthe inversions.Heuts (19 8) has been able to correlate certainphysiological properties (temperature and humidity require ments) in D. paeudoobscura with specific inversion types.Evidence linking the inversion types with specific metabolicfunctions is, however, lacking in the literature.There is considerable literature, however, corre lating point mutations and specific metabolic patternsin other species of Drosophila.Nakamara, ejt al (1953)have studied amino acid metabolism in the four metamorphicstages of zygote maturation in lethal strains of an attachedX strain of D. melanogaster.Nakamara, Imaizumi, andTakanami (1952) have shown that valine, isoleucine and lysineare stable quantitatively while the concentration of glutamicacid decreases during the early development of the zygoteof- D. virilis.During the embryological development ofD. melanomaster the total ninhydrin positive materialreaches a minimum shortly before, hatching and then increases(Van der Crone-Gloor, 1959).Nakamara, Imaizumi and Kitazume(1951a) and Benz (1955) also have been able to show bymeans of paper chromatography that the concentration of
6amino acids and peptides changes during the development ofD. melanogaster.Castiglioni (1953) has chromatographedfor fluorescent substances in different eye pigment mutantsof D. melanogaster and found that there is a close corre lation between the amount of pigment granules in histological sections and the intensity of fluorescence.The heteroaygotes were intermediate between the homozygotesfrom which they were derived.Tondo and Cordeiro (1956)suggest that the electrophoretic properties of componentsof the red eye pigment in D. willistoni may be undergenetic control as these six components differ only intheir physico-chemical properties.Danneel and Zimmerman«(1954) have shown by paper chromatographic analysis ofpupae and imagoes that tryptophan metabolism in vermilioneye color mutants varies with the genotype.Hadorn andMitchell (1950) also working with eye pigment mutants haveshown differences in amino acid metabolism and fluorescentcompounds during the metamorphic stages of D. melanogaater.Beckman and Johnson (19&3) have studied variationsof leucine aminopeptidase (LAP) in D. melanogaster.Usingpupal homogenates, these investigators have demonstratedthe presence of two electrophoretic variants from differentstocks.Thehybrids resulting from a cross betweenthese two strains showed the presence of both variants.Beckman and Johnson (1963) also were able to demonstrate
the presence of genetic variants of phosphatase in larvalhomogenates of different D. melanogaster stocks.Mixturesof homogenates of these different stocks contained bothphosphatase variants.Kikkawa (1963) has demonstrated the presence offast and alow moving amylase components in D. vlrilis.Zymograms ofhybrids between D. virills containing theslow moving component and D. novamexicana containing thefast moving component show both the slow and fast movingcomponents, indicating that each amylase gene producesits own product.Kikkawa (1965) has also demonstratedthe presence of fast and slow moving amylase componentsin D. similans.These two components are controlled byallelic and co-dominant genes on chromosome nujnber two.Doane (1966) has also studied *C-amylase components by meansof disc electrophoresis and has been able to demonstrate therpresence of eight different banding patterns ii\ variousstrains of D. melanogaster.Heterozygotes produced fromthese various strains show additive effects of the allelesfrom each parent indicating that no hybrid enzymes areproduced and that the amylases are monomers.
MATERIALS AND METHODSStocks of the three inversion races of D, paeudoobscura. Standard, Arrowhead and Chiricahua, were obtainedfrom the laboratory of Dr. Theodosius Dobzhansky.Dr.Dobzhansky obtained these three stocks from the samegeographical location* Pinion Plats, Mt. San Jacinto,California, and maintained them in the laboratory formany generations.As far as can be determined the strainsare isogenic.EXPERIMENTAL POPULATIONSEstablishment and Screening of PopulationsExperimental populations of each of the three inver sion races were established by mating virgin females fromeacu of the inversion.races to males from the same inversionrace.The descendents of these matings were used in allsubsequent experiments.These populations were carefullyscreened for ten generations to ascertain whether or notany morphological mutations were present.No detectablemorphological mutations were observed during this initialscreening period.8
9Following the Initial screening, each succeedinggeneration was screened at two day intervals for thepresence of mutations.Populations containing suspectindividuals were discarded.The purity of each inversion race was acertained"by periodic random sampling of each generation throughthird instar larva salivary gland smears.Inversion heterozygotes were produced by matingthree females and two males from the various races.Onlythe P individuals were utilized in the variousexperiments.Growth ConditionsAll populations were grown on cornmeal-molassesagar medium (Strickberger, 1962) in 8 ounce flint glassbottles plugged with gauze wrapped cotton stoppers.Iden tical populations were maintained in Precision BODtemperature control chambers at both 162 C 0.5 C.C 0.5C andStock populations raised at 25 C weretransferred to new medium at four week intervals whilestock populations raised at 16 C were transferred to newmedium at six week intervals.Populations needed for thevarious experimental procedures were started wheneverthey were required.BOD boxes and all work surfaces weretreated weekly with a 1:1 mixture of benzyl benzoate andmineral oil to prevent infestation with mites.
10AMINO ACID ANALYSISQualitative analysis of the free amino acids in thevarious metamorphic stages of both inversion horaozygoteaand inversion heterozygotes was accomplished by extractingthe free amino acids then separating and identifying themby means of thin layer chromatog
Numerous qualitative amino acid differences were also found. Of the twenty amino acids commonly occurring in proteins only alanine and tyrosine were not detected in any metamorphic stage of any genotype at either 16 G or 25 G« Th remaining eighteen commonly occurring amino acids oocurred in three general categories. Four amino
amino acid recommendations based on amino acid levels, and functional vitamin and mineral cofactor recommendations based on amino acid metabolism. These nutrient need suggestions are synthesized depending on the patients’ amino acid results, taking into account the age/gender of the patient and the severity of abnormal findings.
SSIEM classification of Inborn Errors of Metabolism 2011 . Disease group / disease . ICD10 : OMIM : 1. Disorders of amino acid and peptide metabolism ; 1.1. Urea cycle disorders and inherited hyperammonaemias . Other disorders of branched-chain amino acid metabolism 1.4. Disorders of phenylalanine or tyrosi
The amino acid degradation The first step in degradation of many standard amino acids is the removal of the α-amino group, i.e. deamination or transamination. The product is mostly the corresponding 2-oxoacid (α-ketoacid) and α-amino group is released as ammonia or ammonium ion. Direct deamination of amino acids
Introduction to amino acid metabolism Overview The body has a small pool of free amino acids. The pool is dynamic, and is . require dietary sources of these amino acids. In general, the synthesis pathways for the essential amino acids are complex, and involve a large number of reactions.
BIOMOLECULES AMINO ACIDS AND PROTEINS SEM-5, CC-12 PART-5, PPT-25 Peptides II Dr. Kalyan Kumar Mandal Associate Professor St. Paul's C. M. College Kolkata. Peptides II Contents 1. The Primary Structure of Peptide/Determination of Peptide sequence 2. Hydrolysis of Peptide to Amino Acids 3. Separation, Identification and Quantification of Amino .
The 3 general reactions Removal of the NH 3 group Removal of the Carboxyl group Transfer of Methyl groups Specific pathways for amino acid carbon metabolism One carbon metabolism Metabolic processing of amino acid nitrogen The Urea Cycle Phosphocreatine, an i mportant energy reservoir I. OVERVIEW
Titration of amino acid: When an amino acid is dissolved in water it exists predominantly in the . isoelectric form. Amino acid is an . amphoteric. compound It act as either an acid or a base: Upon titration with acid it acts as a BASE (accept a proton). Upon titration with base it acts as an ACID (donate a proton)
Trading A-B-C Patterns . Nick Radge . Many trend trading techniques rely on a breakout of price, that is, price continuing to move in the direction of the trend with uninterrupted momentum. However, price tends to ebb and flow back and forth within the larger trend which can in turn offer up other low risk entry points that are not as recognizable as a pattern or resistance breakout. Then .
