A method to estimate the frequency of chromosomal rearrangements induced by CRISPR/Cas9 multiplexing in Drosophila

Using CRISPR/Cas9 to simultaneously induce mutations in two or more target genes, commonly referred to as multiplexing, may result in chromosomal rearrangements such as inversions or translocations. While this may be undesirable in some contexts, the ability to recover chromosomal rearrangements targeted to specific sites in the genome is potentially a powerful tool. Before developing such tools, however, it is first important to measure the frequency with which chromosome rearrangements are induced by CRISPR/Cas9 multiplexing. To do this, we have developed a self-selecting screening system using a Drosophila line that carries an autosomal pericentric inversion in what is known as the autosynaptic form. All progeny of normal females crossed to males of this autosynaptic stock are lethal due to excessive aneuploidy. If an inversion is induced within the female germline, and if it is analogous to the inversion in the male autosynaptic line, then it is possible to recover progeny in which aneuploidy is reduced and viability is restored. Using this self-selection method, we screened 130 females and recovered one new autosynaptic element. Salivary gland polytene chromosome analysis, PCR, and sequencing confirmed the recovery of a breakpoint induced precisely between the two sgRNA target sites. Overall, we demonstrate that CRISPR/Cas9 multiplexing can induce chromosomal rearrangements in Drosophila. Also, in using this particular system, the recovery of chromosomal rearrangements was not a high frequency event.

Polytene chromosome relationships of five species of the Anopheles dirus complex in Thailand

Photographic maps and rearrangements of each salivary gland polytene chromosome arm of Anopheles nemophilous (species F) and of An. dirus species A, B, C, and D of the Dirus group from natural populations in Thailand are presented. Structural conformation of heterokaryotypes and comparison of chromosome banding sequences reveal 10 paracentric inversions. The data on fixed inversion of 3Rb and inversion polymorphism of the X chromosome shared by these species were used to construct a phylogeny of the five members of the An. dirus complex, thereby outlining their patterns of speciation through chromosomal rearrangements.Key words: polytene chromosome rearrangements, Anopheles dirus, phylogeny.

A mid-arm interchange as a potential reproductive isolating mechanism in the medically important Simulium neavei group (Diptera: Simuliidae)

The Simulium neavei group is medically important as a vector of human onchocerciasis and is unique among blackflies because of the attachment to freshwater crabs during parts of the larval and pupal life cycle stages. Detailed larval salivary gland polytene chromosome maps are presented for two taxa designated S. neavei Amani form A and S. neavei Amani form B, which are probably synonymous with the previously described morphospecies, S. nyasalandicum and S. woodi, respectively. Simulium neavei Amani form B differs from S. neavei Amani form A by three paracentric inversions, a large deletion and a chromocentre, while the latter taxon differs from the former one by a mid-arm interchange. The fixed mid-arm interchange (1 – tr) between two related taxa is unique among the Simuliidae. Detailed banding analysis of S. neavei Amani form B shows that, relative to S. neavei Amani form A, sections 16a, 16b, and 17c of the short arm of chromosome one (IS) have been inserted into section 69/70 of the short arm of chromosome three (IIIS). Assignment of directionality for this interchange is inferred because of the near universality in the Simuliidae of a chromosome III long arm to short arm ratio of 2:1. We propose that the mid-arm interchange acted as a primary isolating mechanism because of the segregational load on F1 heterozygous interchange progeny. The magnitude of the segregational load would be dependent on the frequency of pairing and crossing-over between the transposed interstitial segment and its original counterpart in the chromosome complement. Nonpairing in the transposed region and normal random segregation would presumably lead to a 50% of gametes carrying the duplicated or deficient transposed segment, resulting in a segregation load of 50%. However, crossing-over in the paired interstitial region may greatly increase segregational load as result of multivalent formation at metaphase one (MI) and the generation of dicentric bridge(s) and acentric fragment(s) upon cell division. Such meiotic disjunction irregularities could lead to meiotic arrest, gametic loss and (or) cell restitution, and the eventual formation of polyploid (lethal) zygotes.Key words: mid-arm interchange, speciation, Simulium neavei group, cytotaxonomy, vector biology.

Cytotaxonomy of the Simulium damnosum complex from central and northeastern Tanzania

A cytotaxonomic study of the medically important insect vector, Simulium damnosum s.l., revealed the presence of seven and possibly eight distinct taxa from central and northeastern Tanzania. Larval salivary gland polytene chromosome maps are presented for the first time for five cytotypes and one sibling species, which include the Nkusi form, the Sanje form, the Kisiwani form, Ketaketa C1 and C2, and the Kibwezi form. Inversion disequilibrium in males of the Kibwezi form indicate population substructuring is occurring and that this population may be in the early stages of speciation. Adults of the sibling species S. damnosum Kibwezi form and cytotype of the S. damnosum Nkusi form were identified using Malpighian tubule polytene chromosomes. The taxonomic status of the populations under study are discussed in relation to previously published papers and unpublished reports. Dimorphisms for centromere band enhancement occur on all three polytene chromosomes of the complement. The same centromere band can be polymorphic, sex linked, fixed, or lost in various cytotypes. In constructing a partial phylogeny, a hypothetical intermediate is proposed to account for the diverse fate of these centromere band dimorphisms and other inversion polymorphisms in different members of this nearly pan African complex. This pattern of chromosome restructuring is consistent with that seen for other species complexes within the Simuliidae.Key words: Simulium damnosum complex, Tanzania, cytotaxonomy, phylogenetics, and vector biology.

Expression of a Drosophila melanogaster acetylcholine receptor-related gene in the central nervous system.

We isolated Drosophila melanogaster genomic sequences with nucleotide and amino acid sequence homology to subunits of vertebrate acetylcholine receptor by hybridization with a Torpedo acetylcholine receptor subunit cDNA probe. Five introns are present in the portion of the Drosophila gene encoding the unprocessed protein and are positionally conserved relative to the human acetylcholine receptor alpha-subunit gene. The Drosophila genomic clone hybridized to salivary gland polytene chromosome 3L within region 64B and was termed AChR64B. A 3-kilobase poly(A)-containing transcript complementary to the AChR64B clone was readily detectable by RNA blot hybridizations during midembryogenesis, during metamorphosis, and in newly enclosed adults. AChR64B transcripts were localized to the cellular regions of the central nervous system during embryonic, larval, pupal, and adult stages of development. During metamorphosis, a temporal relationship between the morphogenesis of the optic lobe and expression of AChR64B transcripts was observed.

Expression of a Drosophila melanogaster acetylcholine receptor-related gene in the central nervous system

We isolated Drosophila melanogaster genomic sequences with nucleotide and amino acid sequence homology to subunits of vertebrate acetylcholine receptor by hybridization with a Torpedo acetylcholine receptor subunit cDNA probe. Five introns are present in the portion of the Drosophila gene encoding the unprocessed protein and are positionally conserved relative to the human acetylcholine receptor alpha-subunit gene. The Drosophila genomic clone hybridized to salivary gland polytene chromosome 3L within region 64B and was termed AChR64B. A 3-kilobase poly(A)-containing transcript complementary to the AChR64B clone was readily detectable by RNA blot hybridizations during midembryogenesis, during metamorphosis, and in newly enclosed adults. AChR64B transcripts were localized to the cellular regions of the central nervous system during embryonic, larval, pupal, and adult stages of development. During metamorphosis, a temporal relationship between the morphogenesis of the optic lobe and expression of AChR64B transcripts was observed.

enhancer of seizure: A New Genetic Locus in Drosophila melanogaster Defined by Interactions With Temperature-Sensitive Paralytic Mutations

ABSTRACT Mutations in the enhancer of seizure (e(sei)) locus have been isolated on the basis of their ability to cause temperature-induced paralysis of alleles at the seizure (sei) locus at temperatures at which these mutations ordinarily do not paralyze. This enhancer is specific to the seizure locus and is without effect on other temperature-sensitive paralytic mutants including para, nap, tip-E and shi. This suggests that the enhancer responds specifically to the mechanism of paralysis mediated by the seizure mutations. The e(sei) is a recessive mutation which maps to 39.0 on the left arm of chromosome 3. Deficiency mapping has placed it at 69A4-B5 on the salivary gland polytene chromosome map. When a new enhancer allele was isolated following P-M hybrid dysgenesis, there was a concomitant P-element insertion at 69B. In the absence of seizure mutations, the enhancer mutation causes non-temperature dependent hyperactivity when agitated and interferes with the climbing response. Electrophysiological studies examined the effects of increasing temperature on electrical activity in the adult giant fiber/flight muscle system. Neuronal hyperactivity was seen in both e(sei) and sei single mutant homozygotes, but not in wild type. The hyperactivity was more severe in the sei;e(sei) double mutants. The correlation between the physiological effects and the mutant behavior suggests that both sei and e(sei) cause membrane excitability defects. Since previous work has shown that seizure mutants affect [3H]saxitoxin binding to the voltage-sensitive sodium channel, e(sei) may code for a gene product which interacts with this channel.

VITAL GENES THAT FLANK SEX-LETHAL, AN X-LINKED SEX-DETERMINING GENE OF DROSOPHILA MELANOGASTER

ABSTRACT The X-chromosome:autosome balance in D. melanogaster appears to control both sex determination and dosage compensation through effects on a maternally influenced sex-linked gene called Sex-lethal (Sxl; 1-19.2). To facilitate molecular and genetic analysis of Sxl, we attempted to determine the locations of all ethyl methanesulfonate (EMS)-mutable genes vital to both sexes in the region between 6E1 and 7B1. This area includes approximately 1 cM of the genetic map on each side of Sxl and was reported by C. B. Bridges to contain 26 salivary gland polytene chromosome bands. The region appears rather sparsely populated with genes vital to both sexes, since the 122 recessive lethal mutations we recovered fell into only nine complementation groups. From one to 38 alleles of each gene were recovered. There was a preponderance of embryonic lethals in this area, although the lethal periods of loss-of-function mutations included larval, pupal and adult stages as well. Since the screen required that mutations be recessive and lethal to males, our failure to recover new Sxl alleles was the result expected for a gene with a female-specific function. An attempt was made to identify recessive male-specific lethals in this region, but none were found. Precise map positions were determined for eight of the nine vital genes. An interesting feature of the map is the location of Sxl in the middle of a 0.6- to 0.7-cM interval that appears to be devoid of genes vital to both sexes. The genetic location was determined of breakpoints near Sxl for all available chromosome rearrangements. Sxl is most likely located just to the left of band 7A1. We determined the relationship of our EMS-induced mutations in these nine genes to alleles induced by others. From this we conclude that the various genes appear to differ significantly from each other in their relative sensitivity to mutation by EMS vs. X rays.