Thesis (M.S.)--University of Wisconsin--Madison, 1966. / eContent provider-neutral record in process. Description based on print version record. Bibliography: l. 19-20.

Thèse (Ph. D.)--Université Laval, 2004. / Bibliogr. Publié aussi en version électronique.

Thesis (M.S.)--Ohio University, August, 2005. / Title from PDF t.p. Includes bibliographical references (p. 75-85)

Thesis (M.S.)--University of North Carolina at Greensboro, 2007. / Title from PDF title page screen. Advisor: John Tomkiel; submitted to the Dept. of Biology. Includes bibliographical references (p. 58-62).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 124-153).

The subject of this study is a new dominant suppressor mutation Su(pr) which acts on the purple eye-colour mutant (pr) of Drosophila a melanogaster. The Induction of Su (pr) was originally associated with the synthesis of a compound-2R chromosome In SD72/cn bw females. The suppression of p_ was first observed in combination with a homologous pr bearing compound-2L chromosome. Suppressed-pr flies appeared to have a fully wild eye phenotype. The intention of this study was to determine the chromosomal constitution necessary for Su(pr) induction, and to map the suppressor site. To do this, many compound-2R chromosomes were synthesized from several combinations of standard seconds. It was found that SD72 must be present to produce a suppressing compound-2R. The SD72 second carries a pericentric inversion that results in a duplication of 2L heterochromatIn, and an associated deficiency of 2R heterochromatin in the compound-2R S u (p r) chromosome. Suppression, therefore, Is associated with the pericentric inversion found only on SD72. The role of this segmental aneuploidy was studied by detaching several C(2L)pr: C(2R)SD72/,cn bw suppressed strains such that both arms of the Su(pr) compound autosome were recovered independently and established in standard strains. Suppressing and non-suppressing detachment products were recovered with a frequency that varied according to the compound-2R Su(pr) strain from which they were derived. The chromosome mechanics involved in the process of C(2R)SD72/cn bw formation and subsequent detachment implicates alterations to a segment of proxlmial 2R heterochromatin from SD72 in Su(pr) Induction. Loss of Su(pr) in the detachment process correlates predominantly with deletions generated In 2R heterochromatIn. Recombination mapping relative to the two visible heterochromatic markers, Iight and rolled, revealed that Su(pr) Iies to the left of rolIed. SpectrophotometrIc measurements of eye pigments revealed that suppressed-pr and suppressed-pru bw flies had pigment levels that exceeded the wild type. The lethal allele prc4. was not found to be suppressible. / Science, Faculty of / Zoology, Department of / Graduate

The sex-linked vermilion (v) locus is probably the structural gene for the enzyme tryptophan pyrrolase. Mutations at the locus invariably are recessive and result in a bright-red eye colour phenotype accompanied by a loss of tryptophan pyrrolase activity. Extensive genetic, biochemical and developmental studies of v mutations have shown that the gene is a relatively small cistron controlling the catalytic activity of tryptophan pyrrolase which gives rise to kynure-nine, a brown eye pigment precursor, in the larval fat body during a defined developmental period. Alleles of the locus can be broadly grouped into two classes: 1) spontaneous v mutations, the majority of which are suppressible by mutation at the non-allelic suppressor of sable [su(s)] locus, 2) induced v mutations which are all unsuppressible by su(s) alleles. Alleles of both classes behave nonautonomously during development and all map within the definable limits of the v cistron. This investigation was initiated to recover conditional (temperature-sensitive) v alleles which could be used to study further the regulation of the activity of the v gene during development, and to extend our knowledge of the genetic functioning of the locus. A temperature-sensitive (ts) allele of a known structural gene, affecting the catalytic activity of an assayable enzyme, could also enable a determination of the factors responsible for temperature-sensitivity in Drosophila in terms of changes in the gene product. The temperature-sensitive period (TSP) of a ts mutant in Drosophila is defined as that period during development when exposure to the restrictive temperature commits the organism to a mutant phenotype. With a ts v allele, a correlation can be made between the TSP determined phenotypically and the variation in tryptophan pyrrolase activity during development, and thus contribute to a molecular understanding of the TSP. This study has consisted mainly of the following approaches: 1) mutagenesis and genetic screening to recover ts v alleles, 2) an examination of the phenogenetics of one ts v allele, including fine structure mapping, complementation properties, nonautonomous expression in gynandromorphs, and suppressibility, and a comparison of these properties with those exhibited by some non-ts v mutations, 3) a biochemical analysis of the effect of a ts v mutation on the properties of tryptophan pyrrolase, a deter-mination of the TSP of a ts v allele based on the eye phenotype. Both ts v alleles, v[sup ts1] and v[sup ts2], recovered in this investigation cause a vermilion phenotype if v flies are raised at the restrictive temperature (29°C), whereas v[sup ts] flies raised at the permissive temperature (17° or 22°C) have almost normal eye colour. The activity of tryptophan pyrrolase, extracted from [sup ts1] flies raised at 29°G and 22°C respectively, parallels the temperature-dependent phenotypic properties; enzyme activity is markedly reduced in flies raised at 29°C but is almost normal in flies raised at the permissive temperature. The v[sup ts1] mutation behaves like non-ts,induced v alleles at 29°C in its complementation, suppressibility and nonautonomy. Thus, it fails to complement any other v point mutant, is unsuppressible by su(s)² and is developmentally nonautonomous when present with v* tissue in gynandromorphs raised at 29°C. Since the v[sup ts1] allele is viable when heterozygous with deletions removing the v locus and maps within the v cistron as a point, it is assumed to be a point mutation in the v structural gene. Furthermore, the tryptophan pyrrolase controlled by the v[sup ts1] mutant has different in vitro kinetic and temperature-dependent properties when v[sup ts1] flies are raised at 29 C compared to either wild type or tryptophan pyrrolase extracted from v[sup ts1] flies raised at 22°C. The v[sup ts1] mutant demonstrates different phenotypic and enzyme properties between males and females raised at 29°C; hemizygous males are more mutant in phenotype and have lower tryptophan pyrrolase activity than their homozygous sibs. This result apparently is the reverse of the dosage compensation nor-mally demonstrated by wild type tryptophan pyrrolase in which males with one dose of the v⁺ gene have at least the enzyme activity obtained from females with two doses of the v⁺ gene. How-ever, the TSP for the v[sup ts1] mutant is the same for males and females and falls between the early third instar larva and early pupa stages of development. This period corresponds to the maximum pre-adult activity of tryptophan pyrrolase and also correlates with the formation of kynurenine in the cells of the fat bffidy. These results are discussed in relation to a molecular model explaining the genetic and molecular functioning of the v locus during development. The results are consistent with the hypothesis that v[sup ts] and nonconditional v mutations affect different aspects of active tryptophan pyrrolase structure rather than regulation of the rate of synthesis of the enzyme. Thus, suppressible v mutations affect allosteric or regulatory sites of the enzyme which interact with metabolic and develop-mental cofactors, whereas the nonconditional, unsuppressible, induced v mutations probably affect the catalytic sites of t tryptophan pyrrolase. The ts v mutation, v[sup ts1] , has genetic and biochemical properties which are compatible with an effect on the aggregation of enzyme subunits due to conformational changes during enzyme synthesis at the restrictive temperature. / Science, Faculty of / Zoology, Department of / Graduate

The coding properties of the 3 major valine tRNA isoacceptors of Drosophila melanogaster, the nucleotide sequences of tRNA[sub=Val, sub=3b] and tRNA[sub=Val, sub=4] and the nucleotide sequences of genes for these two tRNAs have been determined. Valyl-tRNA[sub=Val, sub=3a] binds strongly to ribosomes in response to the trinucleotide GUA and to a lesser extent with GUU and GUG. Valyl- tRNA[sub=Val, sub=3b] binds strongly in the presence of GUG and very weakly with the other 3 triplets whereas valyl- tRNA[sub=Val, sub=4] binds strongly in the presence of GUU, GUC, and GUA and weakly with GUG. The nucleotide sequences of tRNA[sub=Val, sub=3b] and tRNA[sub=Val, sub=4] were determined by a combination of techniques. For both tRNAs most of the sequence was determined by the method of Stanley and Vassilenko. The sequences at the 5' and 3'-ends of the molecules were determined by wandering-spot analysis. Regions of the molecules that could not be sequenced by these two techniques were determined by the gel read-off method. The use of tRNA modified with chloroacetaldehyde to overcome problems in sequencing RNA by the gel read-off method caused by secondary structure in the RNA is described. The nucleotide sequence of tRNA[sub=Val, sub=4] is: GUUU[sub=m]⁷CCGUm¹GGUG ѱAGCGGDU (acp³ U)AUCACA1ѱCUGCC[sub=m]UIACAm⁵CGCAGAAGm⁷GCCCCCGGѱC Gm¹ AUCCCGGGCGGAAACACCA. About 50% of the U residues at position 20 are modified to acp³U. One of the C residues at position 48 or 49 is probably modified to m5C. The nucleotide sequence of tRNA[sub=Val, sub=3b] is: GUUUCCGѱAGUGS1 AGCGGDacp³ UAUCACGѱGUGCUUC ACACGCACAAGm⁷- GDCCCCGGTѱCGm¹ AACCC GGGCGGGAACACCA. The C residue at position 48 is probably modified to m⁵C. The observed codon responses of the two tRNAs are discussed in relation to the anticodons found. Val The two tRNA[sub=Val, sub=4] genes of the recombinant plasmid pDt55 were sequenced by the Maxam and Gilbert method. This plasmid hybridizes to the 70BC site on the polytene chromosomes, a major site of tRNA[sub=Val, sub=4] hybridization. The two genes are of opposite polarity and are separated by 525 bp of DNA. The genes have identical sequences, which correspond to that expected from the sequence of tRNA[sub=Val, sub=4]. The nucleotide sequence of the tRNA[sub=Val, sub=3b] gene of recombinant plasmid pDt78R was also determined. This plasmid hybridizes to the 84D site, a major site of tRNA[sub=Val, sub=3b] hybridization. The sequence of the gene corresponds to that expected from the sequence of tRNA[sub=Val, sub=3b]. Comparison of the valine tRNA genes sequenced in this study and those determined by other workers shows that tRNA genes from major sites of tRNA[sub=Val, sub=3b] or tRNA[sub=Val, sub=4] hybridization to polytene chromosomes correspond exactly to the tRNA[sub=Val] sequences while tRNA tRNA[sub=Val] genes from minor sites of tRNA hybridization differ at 4 positions from the sequences expected on the basis of the tRNA sequences. The possible significance of this finding is discussed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Serine tRNAs and their genes in Drosophila melanogaster were characterized. The nucleotide sequences of tRNA₄[sup=Ser] (codon UCG), tRNA₇[sup=Ser] (UCA, UCC, UCU), and tRNA[sub=2b][sup=Ser] (AGC, AGU) were determined. Also, the nucleotide sequences of four tRNA[sup=Ser] genes from the X chromosome isolated in recombinant plasmids were determined. Transfer RNA₄[sup=Ser] and tRNA₇[sup=Ser] differ at only three out of 85 positions, including the "wobble" nucleotide of the anticodon. However, tRNA[sub=2b][sup=Ser] is only 72% homologous with tRNA₄[sup=Ser] (62 out of 85 positions, not counting differences in modification). Major regions of sequence homology (> 5 consecutive positions) are found only in the D arm (21 consecutive positions) and in the TѱC arm (11 consecutive positions). Transfer RNA₄[sup=Ser] and tRNA₇[sup=Ser] are indistinguishable by RNA-DNA hybridization. Both hybridize to the same sites on polytene chromosomes in situ, including the major site at 12DE on the X chromosome, and 23E on chromosome 2 (Hayashi et al. (1980). Chromosoma 76, 65-84.) No other purified tRNA than tRNAs[sub=4,7][sup=Ser] has been shown to hybridize to the X chromosome in Drosophila. Therefore, several X-derived recombinant plasmids hybridizing tRNA[sub=4,7][sup=Ser] (pDt 16, pDt 17R, pDt 27R, and pDt 73; Dunn et al. (1979). Gene ], 197-215.) were analyzed. Based on the results of Southern blotting experi-ments, there appear to be eight tRNA[sup=Ser] genes on the four plasmids (one each on pDt 17R and pDt 73; two on pDt 16; and four on pDt 27R). Thus, the 12DE region contains at least eight tRNA[sup=Ser] genes. Restriction mapping and DNA sequence analysis were performed with pDt 16, pDt 17R, and pDt 73. Based on the tRNA sequences, which differ at three positions, the presumptive DNA sequences encoding tRNA[sub=4] [sup=Ser] and tRNA[sub=7] [sup=Ser] can be represented as 444 or 777 genes. DNA sequence analysis gave surprising Ser results in this respect. Analysis of four tRNA[sup=Ser] genes on the three plasmids S identified two 777 genes matching tRNA₇[sup=Ser] plus "hybrid" 774 and 474 sequences. Further, pDt 16 contains both a 777 and a 774 gene as a direct repeat 400 Ser base pairs apart. Since a 444 gene corresponding to tRNA₄[sup=Ser] must exist, there are at least four different types of closely related serine tRNA genes in the D. melanogaster genome. This observation may have implications concerning the evolution and maintenance of reiterated tRNA genes in eukaryotes. In addition to studies on serine tRNAs and their genes, the nucleotide sequences of Drosophila tRNA₅[sup=Lys] and of a tRNA[sup=Arg] were determined. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

In the present study Drosophila melanogaster was used to define the organization and expression of tRNA genes. The three major Drosophila valine tRNAs were isolated and purified by standard chromatographic techniques. Nucleoside analysis indicated that of these tRNAs only tRNA₄Val contained inosine. All three tRNAsVal contained ribothymidine, therefore they resemble yeast tRNAVal in this regard but not the mammalian tRNAsVal which lack ribothymidine. The purified tRNAs were labelled with ¹ ² ⁵I and used to determine the location of the genes for these tRNAs utilizing the technique of in situ hybridization to salivary gland chromosomes. tRNA₄Val hybridized consistently to one site on the right arm of the second chromosome, 56D, which is close to the site of 5S RNA, 56F. tRNA₃bVal hybridized to two sites, 84D and 92B, both on the right arm of the third chromosome. The labelling of site 84D was approximately twice as heavy as that of 92B. Dr. A. Delaney (unpublished) has shown that approximately 13 genes code for tRNA₃bVal per haploid genome. The in situ hybridization data suggests that the 13 genes are divided such that approximately 8 genes are at site 84D and 5 genes are at site 92B. Evidence to support this supposition is derived from measurements on the amount of tRNA₃bVal in mutant flies deficient or duplicated for site 84D on one of their two homologous third chromosomes. tRNA₃bVal amounts, measured relative to the other tRNAVal isoacceptors decrease 31% in the deficiency and increase 30% in the duplication. These results demon- strate a direct relationship of the amount of tRNA₃bVal to gene dosage because the duplication has 8 extra genes, which is a 30% increase and the deletion has 8 fewer genes, a 30% decrease. Finally, it was shown that the amount of total tRNSVal increased by 17% in the duplication but did not decrease in the deletion. This result demonstrates the amount of valine tRNA is under a type of control in which the amount of total valine tRNA is increased to compensate for the deficiency of a single isoacceptor. Also the coding properties of four tRNASer isoacceptors were determined. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate