Memoria para optar al Título Profesional de Médico Veterinario / El control de la transcripción génica en tejido neural y no neural, permite que los genes que codifican para proteínas de expresión exclusiva, se expresen de ésta forma. En el caso de genes de expresión neuronal-específica, se logra por medio de factores de transcripción que ejercen control positivo (activan la transcripción en tejido neuronal) o negativo (reprimen la transcripción en tejido no neuronal). Dentro de los factores descritos como reguladores negativos de la expresión de genes neuronales en tejido no neuronal se encuentra Represor Element 1 Silencing Transcription Factor (REST), que recluta dos tipos de complejos remodeladores de la cromatina: mSin3 y CoREST. Evidencia in vitro sugiere que éste último es fundamental para que REST ejerza su función represora. En esta memoria se utilizó Drosophila melanogaster como modelo de estudio para evaluar la importancia in vivo de este co-represor por medio de la inhibición de su síntesis mediante RNA interferente (iRNA). El estudio se realizó en el tejido alar de Drosophila melanogaster evaluando las alteraciónes morfológicas y cambios de expresión de genes neuronales y no neuronales para acercarse al estudio de la vía de transducción de CoREST. Los resultados obtenidos sugieren que in vivo, CoREST es fundamental para el desarrollo de venas del ala y la especificación neuronal / Financiamiento: Proyecto NEMO-ICMP07-048-F

The new mutation, synpalpi, which was found in the Xple Stock obtained from the Biological Supply House, Chicago, is so named because of the fusion of the antennae along the midline, all other Xple characteristics being normal except for a slight protrusion of the forehead. In stock cultures the mutant appears in greater numbers from normal Xple crosses than from synpalpi Xple crosses - with a predominance of female synpalpi. Experiments were carried out in order to determine the nature of the mutation, the type of inheritance involved, and the linkage group with which the mutation is associated. The mutant is not a gene mutation but a chromosomal aberration since Mendelian ratios were not observed to occur and since the mutation appeared irregularly but persistantly. A lethal factor is suggested by the small number of flies resulting from synpalpi crosses; the absence or small number of synpalpi in the F¹ of synpalpi crosses but its reappearance in 2-8% of the F² progeny; and by the death of flies in two different stages of development - early and late pupal stages. The association of the lethal factor with the "X" chromosome is assumed because few male synpalpi emerge. A deficiency of a part of the "X" chromosome would explain the death of the males and homozygous females. The possibility of a deficiency of a few bands at the tip of the “X” chromosome was substantiated by a review of the literature on deficient "X" chromosomes. It is believed that synpalpi flies are those deficient flies which do manage to survive and that the fused antennae are caused by the movements of the fly and forcing of fluid to the head in its efforts to extricate itself from the pupa case (its tracheae being attached posteriorly to the case). The Xple stock is very sensitive to variations from the optimum temperature of 25°C. Temperatures of 35°C., 29°C, and 15°C. increased the lethal effect of the deficiency - no synpalpi appearing in the F¹ or F² of temperature-treated cultures. In the test for linkage, the mutant appears to be associated with chromosomes I and III - therefore the mutant is not a simple deficiency of the "X" chromosome alone but appears to involve the third chromosome as well. / Science, Faculty of / Botany, Department of / Zoology, Department of / Graduate

Experimental components analysis (Holling 1966) is used to develop a computer model of the four processes of sexual reproduction: mating, egg production, egg fertilization, and oviposition site selection. A general function of interacting populations is developed, and its application to mating and oviposition site selection is discussed. Data from the literature on mating are used to estimate parameter values for this function. A model of egg production and egg fertilization is developed from an experimental study of the vg strain of Drosophila melanogaster. Egg production is a complex process consisting of four components affecting individual ovarioles: ovariole activation, ovariole production, vitellegenesis, and ovariole deactivation. Threshold effects are found to exist for all four components. Egg fertilization is a simple process involving number of sperm stored and a constant probability of successful fertilization. However, results indicate that both egg fertilization and egg production become more complex beyond the range of treatments used here. Assumptions, not supported by data, are made for the processes of oviposition site selection, aging, mortality, and development. These assumptions are combined with the models of mating, egg production, and egg fertilization into a single tentative model for sexual reproduction. Simulations using this model suggest possible effects of ecological importance: a sigmoid relationship between reproductive rate and density; and a chance in tactics with increasing mortality due to predation. / Science, Faculty of / Zoology, Department of / Graduate

The autonomous behaviour of sex-linked recessive temperature-sensitive lethal mutants in Drosophila melanogaster could be demonstrated by the presence of mosaic patches of tissue hemizygous for the mutant created by loss of a ring X chromosome in cells at the permissive temperature (21.5°C) and the absence of such patches at the restrictive temperature (29°C), The presence of patches at both temperatures indicated that the mutant was non-autonomous. Such non-autonomous behaviour might be attributed to the existence of a substance capable of diffusing from the wild type tissue to supplement the mutant tissue. The experiments carried out showed that the presence or absence of mosaic patches could not be directly interpreted as demonstration of autonomous or non-autonomous properties of the mutant. Other factors such as the time of activity of the ts mutant and the type of tissue undergoing ring X loss affected mosaic tissue production. Therefore, the mere presence of mosaic tissue at 29°C could not be used as a criterion for the non-autonomous behaviour of the ts mutants. However, these mutants can be graded according to the degree of autonomy of ts lethality after alterations due to XO survival frequencies, lethal periods, and temperature-sensitive periods have been placed onmosaic frequencies at 29°C. Of the thirteen ts mutants studied, six can he classed as autonomous lethals. The others are equally autonomous as lethals but only in specific tissues, while others do not appear to be as autonomous. In fact, one of these may be considered non-autonomous. / Science, Faculty of / Zoology, Department of / Graduate

Heterochromatin can be described cytologically as those chromosomes or parts of chromosomes which remain heteropycnotic, or dark staining, through most of the cell cycle. Genetically and biochemically heterochromatic regions generally seem to be inert and it has been suggested that many heterochromatic loci are duplicated several times. In micro-organisms, genetic and biochemical analyses have been greatly facilitated by the use of conditional lethals which survive under "permissive" conditions but die under "restrictive" conditions. Temperature-sensitive ethyl methanesulfonate-induced lethal mutations (such mutants result in survival at 22°C but death at 29°C) have previously been used in Drosophila melanogaster for preliminary studies of development. In the present study 8 temperature-sensitive (ts) sterile mutations (males are fertile at 22°C but sterile at 29°C) were induced on the Y chromosome of D. melanogaster. The ts mutants were mapped genetically on the long arm of the Y chromosome and they were found to involve a minimum of 4 different loci. The Y chromosome of D. melanogaster is entirely heterochromatic and it is necessary for male fertility but the exact function of the Y chromosome is uncertain. The recovery of point mutations (ethyl methanesulfonate-induced temperature-sensitive mutations are presumed to be point mutations) on the Y chromosome indicates that there are loci on the Y represented by a single copy. A determination of the specific developmental effects of the ts sterile mutations, was also attempted. By exposing mutant males to a 48 hour period under the restrictive conditions (29°C) and observing their fertility for several days, the stage in the production of mature sperm during which the ts mutants were having an effect, was determined. / Science, Faculty of / Zoology, Department of / Graduate

The term, heterochromatin, refers to those portions of a chromosome which stain very darkly early in the division cycle at a time when most of the chromosomal material stains very lightly. After the discovery of the mutagenic effects of X-rays, large numbers of mutants were recovered in Drosophila. These mutants were genetically mapped but none of them seemed to be located in the heterochromatic regions of the chromosomes. This observation led to the hypothesis that heterochromatin was genetically inert. Since then the problem of the function of heterochromatin has engaged the attention of many different researchers. One of the few mutants known to reside in the proximal heterochromatin of the X chromosome is bobbed. Ritossa and Spiegelman (1965) have presented evidence that bobbed is the nucleolus organizer, the site of ribosomal RKA synthesis. Their results suggest that this is a highly redundant region of the chromosome. If heterochromatin in general is greatly redundant, this would offer one explanation for the apparent lack of genetic loci. A mutant phenotype might result only after large blocks of the chromosome have been deleted. A scheme involving the use of attached-autosomes to select for those third chromosomes which have sustained breaks in the proximal heterochromatin was devised. In general, virgin females were irradiated and attached-third chromosomes were selected for by mating these females to attached-third males. Female progeny carrying these newly synthesized attached-autosomes are, in turn, irradiated and newly reconstituted chromosomes were selected for by mating the females to males carrying normal third chromosomes. Thus, each newly reconstituted chromosome has sustained a number of breaks at different positions in the proximal heterochromatin. Using this procedure, we recovered sixty-six homozygous lethal chromosomes. Fifty-three of these sixty-six lethals may be placed in one of the six complementation groups, two of which appear to be multisite deletions. In addition, these fifty-three lethals appear to be located, by the results of genetic mapping, in the region spanning the centromere. These two pieces of evidence suggest that the method used here does, indeed, select for deletions in the proximal heterochromatin. / Science, Faculty of / Zoology, Department of / Graduate

The monofunctional ethylating agent EMS has been found to induce ts lethal mutations in Drosophila melanoqaster. Temperature sensitive lethals can be induced on both the X and the second chromosomes. The frequencies of ts lethal induction with respect to the estimated number of "single hit" lethal mutations are not significantly different (for the X chromosome 12.5% and for the second, 10.9%). The ts lethals from each chromosome have similar visibilities at high and low temperatures. As with the ts lethals on the X chromosome, Suzuki et al, 1967) those on the second behaved in a manner compatible with the suggestion that Drosophila ts lethals are similar to those found in micro-organisms. These mutants, particularly dominant ts lethals, may provide powerful tools for Drosophila genetic investigation. / Science, Faculty of / Zoology, Department of / Graduate

The experiments reported in the present dissertation were undertaken to obtain further evidence for the possible roles of protein, RNA and DNA macromolecules in radiation-mutagenesis in Drosophila melanogaster. Several antibiotics were tested for their modifying effects on the frequency of radiation-induced sex-linked recessive lethals. Pre-radiation treatment with actinomycin D significantly reduces the frequency of induced mutations in germ cell stages assumed to include spermatids and spermatocytes. These results are consistent with the hypothesis of a role of proteins in the stabilization (repair) of radiation-induced premutational lesions. Puromycin, a specific inhibitor of protein synthesis is ineffective in the modification of induced mutation frequencies in Drosophila melanogaster. Mitomycin C, is itself a potent mutagen in all germ cell stages, peak mutagenicity occurring in spermatid stages. In combination with ɣ-rays, mitomycin C shows an overall additivity of effect. Mutation frequencies due to mitomycin C are not altered by pre- or concurrent treatment with actinomycin D. This may indicate a different mechanism for mutagenesis by mitomycin C and radiation. / Science, Faculty of / Zoology, Department of / Graduate

Polyhomeotic (ph) is a developmentally important gene in Drosophila melanogaster which has been genetically characterized and recently cloned. ph is genetically and molecularly complex and has a strong maternal effect. Analysis of null or amorphic alleles reveal phenotypic effects that include embryonic lethality, cell death of the ventral epithelium, homeotic transformations, and alteration in the pattern of axon pathways. Two independent point mutations are required to produce a ph null allele. I have shown that the ph locus contains two, large, highly conserved, tandem repeats that are both transcribed. I have identified transcripts that are altered in ph mutants and that are developmentally regulated. Fourteen cDNA's have been isolated, and mapped. Northern and Southern blot analysis, and comparisons between cDNA and genomic restriction maps shows that the cDNAs represent at least 4 different transcripts that include distinct products of both repeats as well as non-repeated sequence. Both the genetic behavior and molecular organization of the ph locus are unique in Drosophila. / Science, Faculty of / Zoology, Department of / Graduate

Multigene families have posed an acute problem for evolutionary biologists ever since the revelation that many families exhibit unexpected sequence homogeneity within and between individuals of a species. A family that is shared between several species, in contrast, often reveals substantial heterogeneity between them. This cohesive and species-specific pattern of variation, which disengages from the classical mode of random genetic drift and selection, has been formally described as Molecular Drive (Dover, 1982). Based on initial observations (Cribbs 1982), the tRNA₄Ser and tRUA₇Ser genes on the X-chromosome of Drosophila melaaogaster also showed intriguing characteristics reminiscent of Molecular Drive. However, in this unusual case, the coevolution process would not only encompass the individuals within a family, but would also ensnare members from a different family. This thesis is an in depth study on the concerted evolution of both gene families and provides evidence consistent with the view that they are undergoing Molecular Drive. Eight tRNA₄,₇Ser genes have been cloned from bands 12DE on the X-chromosome of D metanogaster by molecular walking. There are two tRNA₄Ser and two tRNA₇Ser genes that contain sequences expected from their known tRNAs (Cribbs et. al., 1987a). Of the 86 nucleotides, they only differ from each other at positions 16, 34 and 77 (non-standard numbering, see Sprinzl et al., 1987). The difference at position 34 corresponds to the anticodon and accounts for their difference in codon recognition. These genes have been designated as either 444 or 777 genes, based solely on the three diagnostic differences. However, there is also a single 474 and two 774 genes, which are recombinant structures of the bona fide genes. The remaining gene, 444*, has the three nucleotides diagnostic of tRNA₄Ser but contains a mutation at the tip of the extra arm. Thus collectively, the entire caste of tRNA₄,₇Ser genes at 12DE forms a graded series of transitional states, bridging the narrow sequence variability between true tRNA4Ser and tRNA7Ser. Flanking sequences of these hybrid and the 444* genes show segmental homologies related to both the 444 and 777 genes within the cluster, again a strong indication that both gene types are undergoing concerted evolution. Examination of selected genes from two distantly related sibling species, D, erecta and D. yakuba, shows their equivalent flanking sequences have diverged from those of melanogaster. As expected, the base changes in these species, often occurring as clusters, are also non-random and appear to have been propagated to certain respective members to maintain a species-specific and cohesive pattern of variation consistent with Molecular Drive. One possible mode of spreading sequence variation and creating the hybrid genes in the process could involve an initial stage of asymmetric pairing between 444 and 777 DNA. To examine this possibility, a tRNAArg gene cluster also from 12DE was conveniently exploited as independent "monitors". This family shows fluctuations in the number of genes among the different species and strains (Newton, unpublished), which could also be explained by asymmetric pairing of DNA followed by unequal exchange. Thus, even though the tRNAArg and tRNA₄,₇Ser genes have embarked on different evolutionary pathways, both phenomena may be explained by their common susceptibility to local asymmetric pairing of DNA. / Science, Faculty of / Zoology, Department of / Graduate