The genetics of a new mutable allele at the white locus in Drosophila melanogaster

Summary The genetics of a third case of high mutation frequency at the white locus in Drosophila melanogaster has been analyzed. The new mutable allele, w ^+u, mutates from a wild-type to a white-eyed phenotype in both males and females. The mutational event is 1) premeiotic, 2) not associated with crossingover, 3) sensitive to genetic modification, and 4) restricted to germinal tissue. The only mutants produced by w ^+u are deletions of the white locus. These deficiencies include subsites 4 and 5 of the white locus, but are cytologically unobservable. The mutable allele itself maps to subsite 4.The mutational properties of w ^+u are unlike those of the other highly mutable white alleles which have been interpreted in terms of phage-like controlling elements. Rather, the properties of w ^+u favor a model based on the premature termination of chromosome replication near the terminus of a replicon which leads to a chromosome deficient for the material between the point of premature termination and the end of the replicon.Supported by NIH predoctoral traineeship GM-150 and by NIH research grant GM-07428 to Dr. W. K. Baker.From a dissertation submitted to the Division of Biological Sciences of The University of Chicago in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Regulation of white locus expression: the structure of mutant alleles at the white locus of Drosophila melanogaster

We have analyzed the structures of 19 mutant alleles at the white locus of Drosophila melanogaster. Thirteen of the mutant alleles in our selected sample arose spontaneously, and of these, seven are associated with insertions of non-white-region DNA sequence elements. Several lines of evidence strongly suggest that these insertions are responsible for their associated mutant alleles, and further suggest that most or all of these insertions are transposons. Moreover, the white locus DNA sequences can be divided into two nonoverlapping domains on the basis of the properties of the two domains as mutational targets. One of these domains behaves, in this regard, in the manner expected of functional coding sequences, whereas the other does not. We propose a model for the nature and function of the presumptive noncoding white locus genetic elements. The two domains of the white locus defined by our studies are approximately coextensive with the functionally distinct subintervals of the locus defined by previous genetic analysis. Lastly, our results strongly suggest that the dominant, mutable wDZL allele results from the insertion of a transposon outside of, but near, the white locus. This putative transposon apparently carries genetic elements that act at a distance to repress expression of the white locus.     

A mutable white-inversion in Drosophila melanogaster

Summary From a zeste mutant stock with a mutable white locus a new mutant (z w ^w ) was isolated. It has a white-eyed phenotype and a short X-chromosome inversion (In(1)w ^w ) which extends from salivary chromosome bands 3B2-C1 to 4B4-C1. In giant chromosomes of heterozygotes the inversion is unusually tightly paired. Probably because of this intimate pairing the recombination frequencies for regions near the inversion are not decreased in comparison to those for structurally normal chromosomes. The inversion chromosome is mutable. The mutations which arise have pigmented eyes and can be subdivided into two groups. One group is characterized by a re-inversion to normal chromosome structure. The mutability of the white locus appears to be independent of the inversion and reinversion. The process of reinversion is discussed.     

Interactions among modifiers of retrotransposon-induced alleles of the white locus of Drosophila melanogaster

Mutations in five loci that modify the phenotype of whiteapricot (wa), caused by the retrotransposon, copia, were examined in two-way combinations to determine whether their effects were additive or epistatic. All two-way combinations of mutations in these five loci, mottler of white (mw), suppressor of forked (su(f], suppressor of white apricot (su(wa], Enhancer of whiteapricot, (E(wa] and Darkener of apricot (Doa), are additive in their effects on wa, implying that each second-site modifier locus affects a different process. Three other copia-induced mutations, HwUa, whd81b25 and ctns were also examined for responsiveness to mutations in these modifier loci. None clearly responded. Mutations associated with B104 insertions, including Gl, vgni, ctn and wric were also examined for responsiveness to mw mutations, which have specificity for this element as well. Both vgni and wric respond to mutations in mw. The former interaction demonstrates that mw is capable of interacting with B104 elements in loci other than white. The significance of the results with respect to the nature of second-site modifier loci is discussed.     

Superunstable alleles at the cut locus in Drosophila melanogaster

Summary This is a detailed study of the reversions of the ct ^MR2 allele putatively carrying á mobile element (MR-transposon) in the cut locus. Stable, unstable and superunstable revertants have been identified. Besides, a series of multiple unstable visible and lethal ct mutations derived from the ct ^MR2 allele have been obtained. They are shown to include supermutable alleles. The results suggest that the MR-transposon is connected with at least three functions: excision; change of orientation; and change of position within the cut locus, these functions being disturbed in different ways in different unstable ct ⁺ and ct alleles. In some cases the mutant transitions are somehow strongly stimulated leading to superinstability, reaching the rate of 0.5.     

DNA sequence of the white locus of Drosophila melanogaster

The DNA sequence of the white locus of Drosophila melanogaster is presented. This 14,100 base-pair sequence includes the region of the locus required for wild-type levels of expression and control of expression. We also report the sequence of a complementary DNA clone which established the position of the 3' end of the white RNA on this genomic sequence. The probable exon-intron structure of the gene has been predicted from the DNA sequence of the regions known to be represented in the RNA. The amino acid sequence of the protein which would be produced by translation of this RNA suggests that the white locus gene product may be a membrane protein. The DNA sequence rearrangements associated with seven insertion mutants (white-dominant-zeste-like (wDZL), white-spotted (wsp), white-honey (wh), white-zeste-mottled (wzm), white-apricot (wa), white-buff (wbf) and white-hd81b11 (whd81b11)), one deletion mutant (white-spotted 4 (wsp4)) and one internal duplication mutant (white-ivory (wi)) have been determined and positioned on the wild-type sequence. The positions of these insertions and those of previously characterized insertions associated with six other mutations suggest that some insertions within an intron may still allow the production of correctly spliced RNA, but affect the amount, and correspondingly the expression of the w locus.     

A dosage-sensitive modifier of retrotransposon-induced alleles of the Drosophila white locus.

The apricot allele of the white locus results from the insertion of the retrotransposon copia. Mutations in a newly discovered locus, the Darkener-of-apricot (Doa), suppress wa and some of its revertants. Of 44 other white alleles tested, only wsp55 is affected by Doa, although, in contrast, it is enhanced by Doa mutations. The Doa locus modulates wa and wsp55 expression as a function of its own dosage. Mutations in Doa are dominant suppressors or enhancers and are recessive lethals. Rare Doa mutant homozygotes escaping lethality demonstrate extreme phenotypic suppression of wa and enhancement of wsp55. RNA from wa is substantially wild-type in structure in escapers, although reduced in quantity.     

Complementation between alleles at the ovarian tumor locus of Drosophila melanogaster

The (ovarian tumor) otu gene resides at 23.2 on the genetic map of the X chromosome and near 7F1 on the cytological map. This germ line-expressed locus behaves as if it encodes a gene product which is required during certain steps in the transformation of oogonia into functional oocytes. On the basis of their ovarian morphologies 17 ethyl methane sulfonate (EMS)-induced mutants have been distributed among three developmental classes as follows: quiescent (eight), oncogenic (four), and differentiated (five). The otu13 and otu14 alleles interact to yield fertile females, and many other heteroallelic combinations show partial complementation. Since many mutant alleles interact beneficially, the functional product of the otu gene may be a multimer. We conclude, from an analysis of heteroallelic interactions and dosage effects, that the abnormal phenotypes observed are graded consequences of reduced levels of functional gene product and that the minimum concentration required for development increases as oogenesis proceeds.     

Ovarian pathologies generated by various alleles of the otu locus in Drosophila melanogaster

A comparative cytological study was made of oogenesis in flies carrying various mutant alleles of the female sterile gene otu. It resides at 22.7 on the genetic map and within subdivision 7F of the cytological map of the X-chromosome. Each of the five ethyl methane sulfonate-induced mutations observed falls into one of three classes. In class 1, most mutant ovarioles lack germ cells; in class 2, most mutant ovarioles contain tumorous chambers; and in class 3 mutants, chambers occur that possess defective oocytes. The otu² allele belongs to class 1; otu¹ to class 2; and otu³, otu⁴, and otu⁵ to class 3. The mutations have no effects upon female viability or upon the viability and fertility of hemizygous males. Heterozygous females are fertile and have cytologically normal ovaries. In otu⁵ homozygotes, all ovarioles contain egg chambers, but oogenesis is prematurely terminated to produce a pseudo-stage 12 oocyte. Ovarioles from otu³ and from otu⁴ homozygotes contain both ovarian tumors and oocytes. Pseudonurse cells (PNC), which are cystocytes that have stopped dividing and have entered the nurse cell mode of development, are also abundant. PNCs contain polytene chromosomes. Since the homologs are paired, each nucleus has the haploid number of chromosomes. In chambers lacking an oocyte, the number of PNCs is less than the normal number of nurse cells. In chambers containing an oocyte, the number of accompanying nurse cells may be 15, or above or below normal. In vitellogenic chambers, the chromosomes in the nurse cells connected directly to the oocyte are more expanded than those in more distant nurse cells. The KA14 deficiency lacks the plus allele of otu. KA14 heterozygotes are fertile and have cytologically normal ovaries. When females carry KA14 and otu¹, otu³, otu⁴, or otu⁵, 80% of their ovarioles are agametic. When females carry otu² and one of the other mutant alleles, the ovarioles proceed further in development. So otu² produces a product that has a beneficial effect on the test allele. When two different otu alleles are combined in a single fly, the phenotype of the hybrid ovary usually most resembles that of the ovary homozygous for the “stronger” allele (the otu mutant that allows oogenesis to proceed farthest). The results indicate that the product of the otu⁺ locus functions at least three different times during oogenesis; first to permit oogonia to proliferate, second to control the division and differentiation of germarial cystocytes, and third to facilitate the normal growth of the ooplasm. The gene product appears to be required in higher concentrations at each developmental period. The lesions produced by the mutations are thought to interfere with the stability or functioning of the gene product, and the ovarian phenotype produced by a given genotype depends upon the concentration of functional gene product available to the germ cells.     

Variation between electrophoretically identical alleles at the alcohol dehydrogenase locus in Drosophila melanogaster

A new variant of alcohol dehydrogenase (ADH 7lk) was found in a laboratory stock of Drosophila melanogaster. ADH in this stock had the same electrophoretic mobility as the F variant both on acrylamide and on agar. Activity levels were similar to the levels in F flies at temperature between 15 and 25 C. But while ADH F enzyme is inactivated rapidly at 40 C, ADH 7lk is still active. Also, ADH S is not inactivated at this temperature, but has a far lower activity per fly than ADH 7lk. Genetic analysis showed that the new variant is an allele of the Adh locus.     

Fertile heteroallelic combinations of mutant alleles of the otu locus of Drosophila melanogaster

Summary This paper describes the ovarian pathologies observed when 108 different heteroallelic combinations were made involving 17 independent mutations at the ovarian tumor (otu) locus. Most of the mutant phenotypes can be explained as graded responses by individual germ cells to different levels of functionally active otu gene product (OGP) synthesized by the mutant cells themselves. The lowest and highest levels of OGP appear to be produced by otu ¹⁰ and otu ¹⁴, respectively. In most heteroallelic ovaries the alleles have additive effects, and hybrid germ cells reach a developmental stage more advanced than the weaker homozygote but less advanced than the stronger homozygote. However, examples of both positive and negative complementation also have been found, and these suggest that the products encoded by different mutant alleles can combine to form dimers or multimers which may be superior or inferior to the homodimers. In flies homozygous for otu ¹¹ most ovarioles contain tumors, but some germ cells are able to develop further than those in otu ¹⁴ homozygotes. This suggests that, while otu ¹¹ produces intermediate levels of OGP, it also produces a second product (which otu ¹⁴ cannot make) that is utilized at the period in oogenesis when development in cells homozygous for otu ¹⁴ is blocked. When otu ¹¹ is combined with any one of eight specific alleles, it allows oocyte/nurse cell syncytia to differentiate that can complete development and undergo embryogenesis, if fertilized. The endopolyploid nurse cells of these hybrids have giant polytene chromosomes, and the presence of GPCs in functionally active, germ-line derived cells provides an interesting new system for experimental study. Analysis of the characteristic ovarian pathologies produced by flies of different genotypes leads to the conclusion that the products of the otu ⁺ gene are utilized during at least six different periods in Drosophila oogenesis.     

The nature of radiation-induced mutations at the white locus of Drosophila melanogaster

X-Ray- and neutron-induced mutations at the white locus of Drosophila melanogaster were used to study the nature of radiation-induced genetic damage. Genetic analysis showed the presence of multi-locus deficiencies in 15 out of 31 X-ray mutants and in 26 out of 35 mutants induced by neutrons. The DNA from 11 X-ray and 4 neutron mutants, which were not multi-locus deficiencies, was analyzed by Southern blot-hybridization. Deletions were observed in 2 X-ray and 1 neutron mutant. In combination with cytogenetic techniques, chromosomal rearrangements affecting the white locus (translocations, inversions, etc.) were identified in 3 X-ray and in 2 neutron mutants. A hot-spot for translocation breakpoints was identified in the left arm of the third chromosome. 5 X-ray mutants, which apparently did not contain large deletions, were subjected to further analysis by the nuclease S1 protection method, after cloning of the white gene. In 4 mutants a small deletion could indeed be detected in this way. Thus it seems that by far the main part of X-ray- and neutron-induced white mutants have arisen through large changes in the white gene, especially deletions.