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Traumatic brain injury (TBI) outcomes vary greatly among individuals, but most of the variation remains unexplained. Using a Drosophila melanogaster TBI model and 178 genetically diverse lines from the Drosophila Genetic Reference Panel (DGRP), we investigated the role that genetic variation plays in determining TBI outcomes. Following injury at 20–27 days old, DGRP lines varied considerably in mortality within 24 h (“early mortality”). Additionally, the disparity in early mortality resulting from injury at 20–27 vs 0–7 days old differed among DGRP lines. These data support a polygenic basis for differences in TBI outcomes, where some gene variants elicit their effects by acting on aging-related processes. Our genome-wide association study of DGRP lines identified associations between single nucleotide polymorphisms in Lissencephaly-1 (Lis-1) and Patronin and early mortality following injury at 20–27 days old. Lis-1 regulates dynein, a microtubule motor required for retrograde transport of many cargoes, and Patronin protects microtubule minus ends against depolymerization. While Patronin mutants did not affect early mortality, Lis-1 compound heterozygotes (Lis-1x/Lis-1y) had increased early mortality following injury at 20–27 or 0–7 days old compared with Lis-1 heterozygotes (Lis-1x/+), and flies that survived 24 h after injury had increased neurodegeneration but an unaltered lifespan, indicating that Lis-1 affects TBI outcomes independently of effects on aging. These data suggest that Lis-1 activity is required in the brain to ameliorate TBI outcomes through effects on axonal transport, microtubule stability, and other microtubule proteins, such as tau, implicated in chronic traumatic encephalopathy, a TBI-associated neurodegenerative disease in humans.

Traumatic brain injury (TBI) pathologies are caused by primary and secondary injuries. Primary injuries result from physical damage to the brain, and secondary injuries arise from cellular responses to primary injuries. A characteristic cellular response is sustained activation of inflammatory pathways commonly mediated by nuclear factor-κB (NF-κB) transcription factors. Using a Drosophila melanogaster TBI model, we previously found that the main proximal transcriptional response to primary injuries is triggered by activation of Toll and Imd innate immune response pathways that engage NF-κB factors Dif and Relish (Rel), respectively. Here, we found by mass spectrometry that Rel protein level increased in fly heads at 4–8 hr after TBI. To investigate the necessity of Rel for secondary injuries, we generated a null allele, Rel(del), by CRISPR/Cas9 editing. When heterozygous but not homozygous, the Rel(del) mutation reduced mortality at 24 hr after TBI and increased the lifespan of injured flies. Additionally, the effect of heterozygosity for Rel(del) on mortality was modulated by genetic background and diet. To identify genes that facilitate effects of Rel(del) on TBI outcomes, we compared genome-wide mRNA expression profiles of uninjured and injured +/+, +/Rel(del), and Rel(del)/Rel(del) flies at 4 hr following TBI. Only a few genes changed expression more than twofold in +/Rel(del) flies relative to +/+ and Rel(del)/Rel(del) flies, and they were not canonical innate immune response genes. Therefore, Rel is necessary for TBI-induced secondary injuries but in complex ways involving Rel gene dose, genetic background, diet, and possibly small changes in expression of innate immune response genes.

Mitochondrial function requires coordinated activities of interacting proteins encoded in both the nuclear and mitochondrial genomes. Nuclear mutations cause human mitochondrial disorders that commonly exhibit unexplained clinical variability (e.g. age of onset and severity)... Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions in vivo are sparse. Here we describe the characterization of a mutation in Drosophila ND23, a nuclear gene encoding a highly conserved subunit of mitochondrial complex 1. This characterization led to the discovery of a mito-nuclear interaction that affects the ND23 mutant phenotype. ND23 mutants exhibit reduced lifespan, neurodegeneration, abnormal mitochondrial morphology, and decreased ATP levels. These phenotypes are similar to those observed in patients with Leigh syndrome, which is caused by mutations in a number of nuclear genes that encode mitochondrial proteins, including the human ortholog of ND23. A key feature of Leigh syndrome, and other mitochondrial disorders, is unexpected and unexplained phenotypic variability. We discovered that the phenotypic severity of ND23 mutations varies depending on the maternally inherited mitochondrial background. Sequence analysis of the relevant mitochondrial genomes identified several variants that are likely candidates for the phenotypic interaction with mutant ND23, including a variant affecting a mitochondrially encoded component of complex I. Thus, our work provides an in vivo demonstration of the phenotypic importance of mito-nuclear interactions in the context of mitochondrial disease.

One hundred years ago, in the first paper in the first edition of GENETICS , Calvin Blackman Bridges provided evidence for the chromosome theory of inheritance, laying the groundwork for much of the genetics research that has followed ([Bridges 1916][1]). As we discuss a paper that is arguably a

Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions in vivo are sparse. Here we describe the characterization of a mutation in Drosophila ND23, a nuclear gene encoding a highly conserved subunit of mitochondrial complex 1. This characterization led to the discovery of a mito-nuclear interaction that affects the ND23 mutant phenotype. ND23 mutants exhibit reduced lifespan, neurodegeneration, abnormal mitochondrial morphology, and decreased ATP levels. These phenotypes are similar to those observed in patients with Leigh syndrome, which is caused by mutations in a number of nuclear genes that encode mitochondrial proteins, including the human ortholog of ND23. A key feature of Leigh syndrome, and other mitochondrial disorders, is unexpected and unexplained phenotypic variability. We discovered that the phenotypic severity of ND23 mutations varies depending on the maternally inherited mitochondrial background. Sequence analysis of the relevant mitochondrial genomes identified several variants that are likely candidates for the phenotypic interaction with mutant ND23, including a variant affecting a mitochondrially encoded component of complex I. Thus, our work provides an in vivo demonstration of the phenotypic importance of mito-nuclear interactions in the context of mitochondrial disease.

A common feature of many neurodegenerative diseases is the accumulation of toxic proteins that disrupt vital cellular functions. Degradative pathways such as autophagy play an important protective role in breaking down misfolded and long-lived proteins. Neurons are particularly vulnerable to defects in these pathways, but many of the details regarding the link between autophagy and neurodegeneration remain unclear. We previously found that temperature-sensitive paralytic mutants in Drosophila are enriched for those exhibiting age-dependent neurodegeneration. Here we show that one of these mutants, comatose (comt), in addition to locomotor defects, displays shortened lifespan and progressive neurodegeneration, including loss of dopaminerigic (DA) neurons. comt encodes N-ethyl-maleimide sensitive fusion protein (NSF1), which has a well-documented role in synaptic transmission. However, the neurodegenerative phenotypes we observe in comt mutants do not appear to depend on defects in synaptic transmission, but rather from their inability to sustain autophagy under stress, due at least in part to a defect in trafficking of lysosomal proteases such as cathepsin-L. Conversely, overexpression of NSF1 rescues α-synuclein-induced toxicity of DA neurons in a model of Parkinson’s disease. Our results demonstrate a neuroprotective role for NSF1 that involves mediation of fusion events crucial for degradative pathways such as autophagy, providing greater understanding of cellular dysfunctions common to several neurodegenerative diseases.

Although evolutionary changes must take place in neural connectivity and synaptic architecture as nervous systems become more complex, we lack understanding of the general principles and specific mechanisms by which these changes occur. Previously, we found that morphology of the larval neuromuscular junction (NMJ) varies extensively among different species of Drosophila but is relatively conserved within a species. To identify specific genes as candidates that might underlie phenotypic differences in NMJ morphology among Drosophila species, we performed a genetic analysis on one of two phenotypic variants we found among 20 natural isolates of Drosophila melanogaster. We discovered genetic polymorphisms for both positive and negative regulators of NMJ growth segregating within the variant line. Focusing on one subline, that displayed NMJ overgrowth, we mapped the phenotype to Mob2 [Monopolar spindle (Mps) one binding protein 2)], a gene encoding a Nuclear Dbf2 (Dumbbell formation 2)-Related (NDR) kinase activator. We confirmed this identification by transformation rescue experiments and showed that presynaptic expression of Mob2 is necessary and sufficient to regulate NMJ growth. Mob2 interacts in a dominant, dose-dependent manner with tricornered but not with warts, to cause NMJ overgrowth, suggesting that Mob2 specifically functions in combination with the former NDR kinase to regulate NMJ development. These results demonstrate the feasibility and utility of identifying genetic variants affecting NMJ morphology in natural populations of Drosophila. These variants can lead to discovery of new genes and molecular mechanisms that regulate NMJ development while also providing new information that can advance our understanding of mechanisms that underlie nervous system evolution.

Neuronal Na+ and K+ channels elicit currents in opposing directions and thus have opposing effects on neuronal excitability. Mutations in genes encoding Na+ or K+ channels often interact genetically, leading to either phenotypic suppression or enhancement for genes with opposing or similar effects on excitability, respectively. For example, the effects of mutations in Shaker (Sh), which encodes a K+ channel subunit, are suppressed by loss-of-function mutations in the Na+ channel structural gene para, but enhanced by loss-of-function mutations in a second K+ channel encoded by eag. Here we identify two novel mutations that suppress the effects of a Sh mutation on behavior and neuronal excitability. We used recombination mapping to localize both mutations to the eag locus, and we used sequence analysis to determine that both mutations are caused by a single amino acid substitution (G297E) in the S2–S3 linker of Eag. Because these novel eag mutations confer opposite phenotypes to eag loss-of-function mutations, we suggest that eagG297E causes an eag gain-of-function phenotype. We hypothesize that the G297E substitution may cause premature, prolonged, or constitutive opening of the Eag channels by favoring the “unlocked” state of the channel.

ONE cannot help but admire a gene as fiendishly clever as Segregation distorter ( Sd ). Not only has it figured out a way to cheat the system, but it is extraordinarily good at what it does. It violates the fundamental principle of Mendelian inheritance regularly and with remarkable potency by

To elucidate the mechanisms regulating expression of para, which encodes the major class of sodium channels in the Drosophila nervous system, we have tried to locate upstream cis-acting regulatory elements by mapping the transcriptional start site and analyzing the region immediately upstream of para in region 14D of the polytene chromosomes. From these studies, we have discovered that the region contains a cluster of neurally expressing genes. Here we report the molecular characterization of the genomic organization of the 14D region and the genes within this region, which are: calnexin (Cnx), actin related protein 14D (Arpl4D), calcineurin A 14D (CnnAl4D), and chromosome associated protein (Cap). The tight clustering of these genes, their neuronal expression patterns, and their potential functions related to expression, modulation, or regulation of sodium channels raise the possibility that these genes represent a functionally related group sharing some coordinate regulatory mechanism.

We cloned genomic DNA corresponding to the Drosophila virilis homologue of para, a gene encoding a sodium channel alpha-subunit, and obtained many partial cDNA clones from embryos and adults. Para protein has been well conserved, and the optional elements at six different sites of alternative splicing in D. melanogaster are present in D. virilis, in addition to one new optional exon. Among 31 different splice-types observed in D. virilis, the stage-specific pattern of alternative splicing seen in D. melanogaster is also conserved. Comparison of genomic DNA sequence revealed three aspects that vary between alternatively and constitutively used exon sequences. Sixteen short blocks (10-75 bp), the only recognizably conserved intron sequence, were disproportionately associated with alternatively used splice sites. Silent site substitutions were found much less frequently in alternative than constitutive exon elements, and the degree of match to the Drosophila splice site consensus tended to be lower at less frequently selected alternative splice junctions. This study shows that the developmentally regulated variability of para products is highly conserved and therefore likely to be of functional significance and suggests that a variety of different sequence-dependent mechanisms may regulate this pattern of alternative splicing.

Segregation Distorter (SD) is a meiotic drive system in D. melanogaster that results in the failure of SD/SD+ males to transmit SD+ homologs owing to the induced dysfunction of spermatids carrying the normal chromosome. Segregation distorter (Sd), the gene primarily responsible for this distorted transmission, is associated with a novel 12-kb restriction fragment containing a tandem duplication of a 5-kb wild-type segment of genomic DNA. When introduced into appropriate genetic backgrounds by germline transformation, this 12-kb fragment causes full levels of distortion and directs the expression of an SD-specific 4-kb transcript. Transformants that have lost part of this segment are unable to cause distortion and do not express the 4-kb transcript. These results identify the tandem duplication as Sd.

Segregation Distorter (SD) is a naturally occurring meiotic drive system comprising at least three distinct loci: Sd, Rsp and E(SD). Heterozygous SD/SD+ males transmit the SD chromosome in vast excess over the normal homolog. The distorted transmission involves the induced dysfunction of the spermatids that receive the SD+ chromosome. In the 220-kb region of DNA that contains the Sd gene, we identified a 5-kb tandem duplication that is uniquely associated with all SD chromosomes, absent in SD+ chromosomes, and detectably altered in Sd revertants. On northern blots, genomic probes from the tandem duplication detect an SD-specific 4-kb transcript in addition to several smaller transcripts present in both SD and SD+. Seven classes of cDNAs derived from these transcripts have been isolated. All of these cDNAs share extensive sequence identity at their 3' ends but differ at their 5' ends. Sequence analysis indicates that these cDNAs potentially encode four distinct, but related, polypeptides. Introduction of the tandem duplication into SD+ flies by germline transformation did not confer the dominant gain-of-function Sd phenotype. This result, taken together with our analysis of the Sd cDNAs, suggests that the duplication is part of a much larger gene that encodes several different polypeptides.

Genes encoding proteins involved in the function of the nervous system can be identified via mutations causing behavioral abnormalities. An example is ether a go-go (eag) in Drosophila melanogaster, which was identified originally as an X-linked mutation that displayed ether-induced leg-shaking behavior. Electrophysiological and genetic evidence suggests that the product of the eag locus is intimately involved in the normal functioning of one or more types of voltage-gated potassium channels. To initiate a molecular analysis of eag we first generated a collection of deletions to pinpoint its cytological location. On the basis of this location, we identified an existing inversion, In(1)sc(29), with one breakpoint at the eag locus and the other in the scute (sc) complex. A genomic library was prepared from In(1)sc(29) and screened with a genomic DNA fragment that spanned the sc breakpoint to isolate DNA from the eag region. Beginning from this starting point over 85 kb of DNA were isolated by chromosome walking. Three additional eag alleles, including two dysgenesis-induced insertion mutations and a γ-ray-induced insertional translocation, were located on the molecular map of the eag locus by Southern blot analysis. The molecular defects associated with these alleles encompass a total of 27 kb within the chromosome walk. A 10-kb transcript derived from this region, which is expressed most abundantly in heads, was identified on Northern blots. Two different eag mutations separated by over 20 kb interrupt the same transcript identifying it as the likely eag message. cDNAs representing a portion of this transcript have been isolated. The genomic DNA sequences from which these cDNAs are derived extend over 37.5 kb, providing a minimum estimate of the size of the eag transcription unit. Ultimately, sequence analysis of these cDNAs should enable us to the identify the eag polypeptide and to elucidate its role in membrane excitability.

Genetic factors are known to contribute to seizure susceptibility, although the long-term effects of these predisposing factors on neuronal viability remain unclear. To examine the consequences of genetic factors conferring increased seizure susceptibility, we surveyed a class of Drosophila mutants that exhibit seizures and paralysis following mechanical stimulation. These bang-sensitive seizure mutants exhibit shortened life spans and age-dependent neurodegeneration. Because the increased seizure susceptibility in these mutants likely results from altered metabolism and since the Na+/K+ ATPase consumes the majority of ATP in neurons, we examined the effect of ATPα mutations in combination with bang-sensitive mutations. We found that double mutants exhibit strikingly reduced life spans and age-dependent uncoordination and inactivity. These results emphasize the importance of proper cellular metabolism in maintaining both the activity and viability of neurons.

The effects of para mutations on behavior and axonal excitability in Drosophila suggested that para specifically affects sodium channels. This hypothesis was confirmed by molecular analysis of the para locus, which demonstrates that the encoded para product is a sodium channel polypeptide. Here we characterize the effects of altered para+ dosage on behavior and axonal excitability, both in an otherwise wild-type background and in combination with two other mutations: napts, which also affects sodium channels, and ShKS133, which specifically affects potassium channels. Whereas it was previously shown that decreased dosage of para+ is unconditionally lethal in a napts background, we find that increased dosage of para+ suppresses napts. Similarly, we find that para hypomorphs or decreased dosage of para+ suppresses ShKS133, whereas increased dosage of para+ enhances ShKS133). The electrophysiological basis for these effects is investigated. Other genes in Drosophila that have sequence homology to sodium channels do not show such dosage effects, which suggests that the para+ product has a function distinct from that of other putative Drosophila sodium channel genes. We conclude that the number of sodium channels present in at least some Drosophila neurons can be affected by changes in para+ gene dosage, and that the level of para+ expression can strongly influence neuronal excitability.

Age-dependent neurodegeneration is a pathological condition found in many metazoans. Despite the biological and medical significance of this condition, the cellular and molecular mechanisms underlying neurodegeneration are poorly understood. The availability of a large collection of mutants exhibiting neurodegeneration will provide a valuable resource to elucidate these mechanisms. We have developed an effective screen for isolating neurodegeneration mutants in Drosophila. This screen is based on the observation that neuronal dysfunction, which leads to observable behavioral phenotypes, is often associated with neurodegeneration. Thus, we used a secondary histological screen to examine a collection of mutants originally isolated on the basis of conditional paralytic phenotypes. Using this strategy, we have identified 15 mutations affecting at least nine loci that cause gross neurodegenerative pathology. Here, we present a genetic, behavioral, and anatomical analysis of vacuous (vacu), the first of these mutants to be characterized, and an overview of other mutants isolated in the screen. vacu is a recessive mutation located cytologically at 85D-E that causes locomotor defects in both larvae and adults as well as neuronal hyperactivity. In addition, vacu exhibits extensive age-dependent neurodegeneration throughout the central nervous system. We also identified mutations in at least eight other loci that showed significant levels of neurodegeneration with a diverse array of neuropathological phenotypes. These results demonstrate the effectiveness of our screen in identifying mutations causing neurodegeneration. Further studies of vacu and the other neurodegenerative mutants isolated should ultimately help dissect the biochemical pathways leading to neurodegeneration.

Post-transcriptional editing of pre-mRNAs through the action of dsRNA adenosine deaminases results in the modification of particular adenosine (A) residues to inosine (I), which can alter the coding potential of the modified transcripts. We describe here three sites in the para transcript, which encodes the major voltage-activated Na+ channel polypeptide in Drosophila, where RNA editing occurs. The occurrence of RNA editing at the three sites was found to be developmentally regulated. Editing at two of these sites was also conserved across species between the D. melanogaster and D. virilis. In each case, a highly conserved region was found in the intron downstream of the editing site and this region was shown to be complementary to the region of the exonic editing site. Thus, editing at these sites would appear to involve a mechanism whereby the edited exon forms a base-paired secondary structure with the distant conserved noncoding sequences located in adjacent downstream introns, similar to the mechanism shown for A-to-I RNA editing of mammalian glutamate receptor subunits (GluRs). For the third site, neither RNA editing nor the predicted RNA secondary structures were evolutionarily conserved. Transcripts from transgenic Drosophila expressing a minimal editing site construct for this site were shown to faithfully undergo RNA editing. These results demonstrate that Na+ channel diversity in Drosophila is increased by RNA editing via a mechanism analogous to that described for transcripts encoding mammalian GluRs.

The segregation distorter (SD) complex is a naturally occurring meiotic drive system with the property that males heterozygous for an SD-bearing chromosome 2 and an SD +-bearing homolog transmit the SD-bearing chromosome almost exclusively. This distorted segregation is the consequence of an induced dysfunction of those sperm that receive the SD + homolog. From previous studies, two loci have been implicated in this phenomenon: the Sd locus which is required to produce distortion, and the Responder (Rsp) locus that is the site at which Sd acts. There are two allelic alternatives of Rsp—sensitive (Rspsens) and insensitive (Rspins); a chromosome carrying Rspins is not distorted by SD. In the present study, the function and location of each of these elements was examined by a genetic and cytological characterization of X-ray-induced mutations at each locus. The results indicate the following: (1) the Rsp locus is located in the proximal heterochromatin of 2R; (2) a deletion for the Rsp locus renders a chromosome insensitive to distortion; (3) the Sd locus is located to the left of pr(2-54.5), in the region from 37D2-D7 to 38A6-B2 of the salivary chromosome map; (4) an SD chromosome deleted for Sd loses its ability to distort; (5) there is another important component of the SD system, E(SD), in or near the proximal heterochromatin of 2L, that behaves as a strong enhancer of distortion. The results of these studies allow a reinterpretation of results from earlier analyses of the SD system and serve to limit the possible mechanisms to account for segregation distortion.

Two classes of X-linked behavioral mutants of Drosophila melanogaster, leg-shaking mutants and bang-sensitive mutants, are suppressed by napts (no action potential, temperature-sensitive), an autosomal temperature-sensitive paralytic mutation. So far, napts is found to suppress thirteen mutations at seven loci, two of which produce leg shaking and five bang-sensitivity. Suppression is recessive, occurs at temperatures permissive for napts, and is indirect and function-specific rather than allele-specific. At restrictive temperatures, napts is known to completely block all nerve activity. Several of the mutants suppressed by napts are shown by neurophysiological experiments to have increased nerve excitability. The physiological defect of these mutants as well as their behavioral defect is suppressed by napts. Thus, suppression occurs within individual neurons at the level of excitable membranes and apparently depends on the reduction in membrane excitability caused by napts even under permissive conditions. We suggest that all mutants suppressed by napts may have related defects leading to enhanced nerve excitability. Genetic interactions of this type help reveal functional relationships between different behavioral mutants and suggest ways of isolating new mutants with altered excitable membranes.

Analysis of X-ray-induced deletions in the Segregation Distorter (SD) chromosome, SD-5, revealed that this chromosome had a gene proximal to lt in the centric heterochromatin of 2L that strongly enhanced the meiotic drive caused by the SD chromosome. This Enhancer of Segregation Distortion [E(SD)] locus had not been characterized in earlier studies of SD chromosomes because it cannot be readily separated by recombination from the Responder (Rsp) locus in the proximal heterochromatin of 2R.—To determine whether E(SD) is a general component of all SD chromosomes and to examine further its effects on distortion, we produced deletions of E(SD) in three additional SD chromosomes. Analysis of these deletions leads to the following conclusions: (1) along with Sd and Rsp, E(SD) is common to all SD chromosomes; (2) the E(SD) allele on each SD chromosome enhances distortion by the same amount, which indicates that allelic variation at the E(SD) locus is not responsible for the different drive strengths seen among SD chromosomes; (3) E(SD) causes very little or no distortion by itself in the absence of Sd; (4) E(SD), like Sd, acts in a dosage-dependent manner; (5) E(SD) exerts its effect in cis or trans to Sd; and (6) if E(SD) + exists, its function is not related to SD.

A combined molecular and genetic analysis of the 87E actin gene (Act87E) in Drosophila melanogaster was undertaken. A clone of Act87E was isolated and characterized. The Act87E transcription unit is 1.57 kb and includes a 556-base intervening sequence in the 5' leader of the gene. The protein-coding region is contiguous and encodes a protein that is greater than 93% identical to the other Drosophila actins. By in situ hybridization with a series of deficiencies that break in 87E, Act87E was localized to a region encompassing one to three faint, polytene chromosome bands. The region between the deficiency endpoints that flank the actin gene was isolated and measures approximately 24-30 kb. The closest proximal deficiency endpoint lies 8-10 kb 5' to the actin gene; the closest distal deficiency endpoint lies 16-20 kb 3' to the actin gene. A single, recessive lethal complementation group lies between the deficiency endpoints that flank the actin gene. An EMS mutagenesis screen produced four additional members of this recessive lethal complementation group. Molecular analysis of the members of this complementation group indicated that two of the newly induced mutations have deletions of approximately 1 kb in a transcribed region 4-5 kb 3' (distal) to the actin gene. This result suggests that the recessive lethal complementation group represents a gene separate from and distal to the actin gene. The mutagenesis screen failed to identify additional recessive lethal complementation groups in the actin gene-containing region. The implications of the failure to identify recessive lethal mutations in the actin gene are discussed in reference to studies of other conserved multigene families and other muscle protein mutations.

Identification of allelic variability at the two major loci (Sd and Rsp) that interact to cause sperm dysfunction in Segregation distorter (SD) males of D. melanogaster has been hampered by the difficulty in separating the elements recombinationally. In addition, small differences in the strength of Sd alleles or sensitivities of Rsp alleles to Sd are difficult to measure against background genetic or environmental variation. Viability effects of the markers used to score progeny classes may also introduce a bias. Removal of Sd and E(SD) from their second chromosome location to create a Dp(2;Y)Sd E(SD) chromosome eliminates these problems, since any combination of Rsp alleles can be easily tested without resorting to recombinational techniques. Further, since these pairs of Rsp alleles are compared in their response to Dp Sd E(SD) in the same individual males, background variation and viability effects can be easily removed to allow finescale resolution of Rsp differences. Tests of all possible pairwise combination of six laboratory chromosomes in this way revealed at least three and possibly four different Rsp allelic classes. In addition, the hierarchical nature of the tests further allowed for determination of the presence of linked suppressors or enhancers of Sd activity. A sample of 11 second chromosomes selected from a group recently isolated from a natural population was also unambiguously ordered as to Rsp allelic status using this approach. The resultant pattern was similar to that obtained for the laboratory chromosomes, except for the not unexpected observation that the natural population apparently harbored more drive suppressors. The pattern of results obtained from these pairwise combinations of Rsp alleles supports the notion that there are no dominance interactions within the group, but that each responds more or less independently to Sd in giving sperm dysfunction.

Segregation distorter (SD) chromosomes are preferentially transmitted to offspring from heterozygous SD/SD + males owing to the induced dysfunction of the SD +-bearing sperm. This phenomenon involves at least two major loci: the Sd locus whose presence is necessary for distortion to occur and the Rsp locus which acts as the site of Sd action. Several additional loci on SD chromosomes enhance distortion.—In a previous study deletions were used to map the Sd locus and to determine some of its properties. We have extended this analysis with the isolation and characterization of 14 new deletions in the Sd region. From our results we conclude (1) SD chromosomes contain a single Sd locus located in region 37D2-6 of the salivary gland chromosome map. Deletion of this locus in any of three SD chromosomes now studied results in complete loss of ability to distort a sensitive chromosome; (2) the reduced male fecundity observed in many homozygous SD or SDi/SDj combinations is due at least in part to the action of the Sd locus. The fecundity of these males can be substantially increased by deletion of one Sd locus. Thus, it is the presence of two doses of Sd rather than the absence of Sd + that produces the lowered male fecundity in SD homozygotes; (3) Sd behaves as a neomorph, whereas Sd +, if it exists at all, is amorphic with respect to segregation distortion; (4) these results support a model in which the Sd product is made in limiting amounts and the interaction of this product with the Rsp locus causes sperm dysfunction. The Sd product appears to act preferentially at Rsps (sensitive-Responder) but may also act at Rspi (insensitive-Responder).