“The Image” of the Regulatory Gene in Experiments with Drosophila

The mutants referred to as facultative dominant lethals were selected in the progeny of gamma-irradiated Drosophila males. The mutant males were viable and fertile, though their crosses with females of the yellow line yielded no daughters. The mutations obtained differed from the common mutations by (1) extremely varying penetrance of F₁ hybrids from crosses with various lines; (2) the uncertain relationships between the mutant and normal alleles; (3) the different expression in somatic and germ cells; (4) the dependence of the expression on the sex of the parent that was the donor of the mutation; (5) the mass morphosis formation and (6) the frequent reversal to the norm. These mutations are assigned to the regulatory group and their specific expression (see above) can be helpful in identifying regulatory gene mutations. We assume that the specific expression of the mutations studied is related to specific properties of the regulatory genes. These properties are as follows: (1) only one out of two homologous regulatory genes is in an active state, (2) in the haploid chromosome set, the regulatory gene is represented by several alleles (cys-alleles); (3) only one allele ensures the regulatory gene activity.     

Gene expression divergence and the origin of hybrid dysfunctions

Hybrids between closely related species are often sterile or inviable as a consequence of failed interactions between alleles from the different species. Most genetic studies have focused on localizing the alleles associated with these failed interactions, but the mechanistic/biochemical nature of the failed interactions is poorly understood. This review discusses recent studies that may contribute to our understanding of these failed interactions. We focus on the possible contribution of failures in gene expression as an important contributor to hybrid dysfunctions. Although regulatory pathways that share elements in highly divergent taxa may contribute to hybrid dysfunction, various studies suggest that misexpression may be disproportionately great in regulatory pathways containing rapidly evolving, particularly male-biased, genes. We describe three systems that have been analyzed recently with respect to global patterns of gene expression in hybrids versus pure species, each in Drosophila. These studies reveal that quantitative misexpression of genes is associated with hybrid dysfunction. Misexpression of genes has been documented in sterile hybrids relative to pure species, and variation in upstream factors may sometimes cause the over- or under-expression of genes resulting in hybrid sterility or inviability. Studying patterns of evolution between species in regulatory pathways, such as spermatogenesis, should help in identifying which genes are more likely to be contributors to hybrid dysfunction. Ultimately, we hope more functional genetic studies will complement our understanding of the genetic disruptions leading to hybrid dysfunctions and their role in the origin of species.     

Cloning and characterization ofDaphnia mitochondrial DNA

Summary The mitochondrial genome ofDaphnia pulex (Crustacea, Cladocera) was cloned as a single fragment into the plasmid vector pUC12. The genome size, estimated from restriction endonuclease fragment lengths, is 15,400±200 base pairs. The GC content, estimated from thermal denaturation studies, is 42%. The positions of 39 cleavage sites were mapped for 14 restriction enzymes. The distribution of these sites within the genome is random (P=0.44). Heterologous hybridizations withDrosophila sylvestris mitochondrial DNA (mtDNA) probes indicate that gene orders withinDaphnia andDrosophila mtDNAs are similar.     

The Reason for the Preservation of High Instability at the yellow Gene in Drosophila melanogaster Strains Isolated from the Natural Population of Uman' during the “Mode for Mutation”

Mobile genetic elements are responsible for most spontaneous mutations in Drosophila melanogaster. The discovered in the 1980s phenomenon of frequent change of the wild-type yellow phenotype for a mutant one, and vice-versa, in strains of D. melanogaster isolated from the Uman' natural population can be, according to our data, explained by repeated inversions and reinversions of the gene regulatory region located between the two copies of the hobo transposon. However, most molecular genetic events accompanying the process can occur without the phenotype change. After several generations, the strains, remaining phenotypically unchanged, can possess different molecular genetic properties with respect to yellow. Using genetically homogenous or isogenic strains for the genetic analysis or for production of the new plant cultivars or animal breeds, geneticists and breeders often face the problem of stability of the strains. In the present study, the mechanism underlying the generation of instability at the yellowlocus of D. melanogaster determined by the hobo-induced genome instability is described.     

Temperature-sensitive yellow mutants of Chlamydomonas reinhardtii

Summary We have isolated and genetically characterized 10 mutants of Chlamydomonas reinhardtii carrying single, mendelian, temperature-sensitive yellow mutations. The mutants have a yellow phenotype at the restrictive temperature (33°C), but have a wildtype phenotype at the permissive temperature (25°C). Based on complementation and recombination tests, the ten mutations include alleles of two previously described yellow loci (y-1 and y-6) and three new yellow loci (y-8, y-9, and y-10). At the restrictive temperature, y-8, y-9, and y-10 are physiologically similar to other yellow mutants. They accumulate small amounts of protochlorophyllide when grown under dim light, but synthesize normal amounts of chlorophyll when grown in the light. Linkage tests indicate that the three new mutations are not linked to each other. y-8 is linked to y-7 on linkage group III, and y-10 is linked to y-5 and y-6 on linkage group I. y-9 is located on linkage group II. We conclude that the control of light-independent protochlorophyllide reduction is complex, involving several genetic loci which are scattered in the genome and which code for gene products able to complement in trans. Temperature-sensitive alleles at several of the yellow loci suggest that the gene products made by these loci are proteins.     

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in GI pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used mAe. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.     

Improved efficiency for T-DNA-mediated transformation and plasmid rescue inArabidopsis thaliana

A vector was constructed for the isolation of gene fusions to thelacZ reporter gene following T-DNA integration into the genome ofArabidopsis thaliana. To facilitate the generation of taggedA. thaliana plants, we established a modified method for high-frequency transformation ofA. thaliana byAgrobacterium tumefaciens. The main modification required was to inhibit the methylation of T-DNA in the transformed calli. Apparently, cytosine residues of thenos-nptII gene used as a selectable marker were methylated, and the expression of this gene was suppressed. Treatment of the calli with the cytosine methylation inhibitor 5-azacytidine led to a dramatic increase (from 3% to 96%) in the regeneration of transformed (kanamycin-resistant) shoots. A total of 150 transgenic plants were isolated, and in 17 of these expression of thelacZ reporter was detected byin situ staining. The T-DNA insert together with flanking plant DNA sequences was cloned intoEscherichia coli by plasmid rescue from some of the T₃ transformants that harbored one copy of the integrated T-DNA. Comparison of the rescued DNA with the corresponding DNA of the transgenic plant showed that most of the rescued plasmids had undergone rearrangements. These rearrangements could be totally avoided if anmcrAB (modified cytosine restriction) mutant ofE. coli was used as the recipient in plasmid rescue.     

Genetic basis of natural variation in body pigmentation in Drosophila melanogaster

Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. Recently, we performed a genome wide association (GWA) analysis of variation in abdominal pigmentation using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). We confirmed the large effects of regulatory variants in bab1, t and e; identified 81 additional candidate genes; and validated 17 candidate genes (out of 28 tested) using RNAi knockdown of gene expression and mutant alleles. However, these analyses are imperfect proxies for the effects of segregating variants. Here, we describe the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. We replicated the effects on pigmentation of 28 genes implicated by the DGRP GWA study, including bab1, t and e and 7 genes previously validated by RNAi and/or mutant analyses. We also identified many additional loci. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.     

A multicopy vector system for genetic studies in<Emphasis Type="Italic"> Mucor circinelloides</Emphasis> and other zygomycetes

Transformation of Mucor circinelloides is routinely achieved by using a plasmid containing the wild-type leuA gene to complement the leucine requirement of an auxotrophic host strain. As is the case for other zygomycetes, the transforming DNA is usually not integrated into the genome of M. circinelloides, but is maintained as an autonomously replicating plasmid. However, even under selective conditions, the plasmid is segregationally unstable, resulting in a rather low number of cells carrying the plasmid. We report here on a new transformation vector based on a dominant selection marker conferring resistance to geneticin, which allows for plasmid maintenance in high copy numbers. The vector was also used to transform Mucor rouxii and Rhizomucor pusillus, and should therefore be a valuable tool for gene expression studies in zygomycetes. The functionality and regulatory properties of the promoter of the M. circinelloides gpd1 gene (which codes for glyceraldehyde-3P-dehydrogenase) were demonstrated in R. pusillus using geneticin selection. In this work, we have also determined the molecular basis of the Leu^- phenotype of the M. circinelloides host strain R7B. The leucine requirement is due to a single point mutation in the leuA gene that results in the replacement of a glutamic acid by a lysine residue.Communicated by E. Cerdà-OlmedoOn 1 January, 2004, the Biotechnological Institute became Bioneer A/S ()     

Function of trans-acting genes of theachaete-scute complex in sensory organ patterning in the mesonotum ofDrosophila

Summary Cell-cell interactions play a fundamental role in the differentiation of nervous elements in constant patterns, both during embryogenesis and imaginal development. In this paper we analyse the role of genes of theachaete-scute andEnhancer of split complexes, plus the genesextramacrochaetae, Notch, Delta, andHairless in the patterning of sensory elements in the mesonotum ofDrosophila. The phenotypes of different alleles of these genes, including lethals in genetic mosaics, reveal their participation in two processes, the singling out from epidermal cells of sensory organ mother cells and their subsequent differentiation. Studies of allelic combinations of different genes lead to a model of the genetic interactions involved in the processes of pattern formation. In this model, theachaete-scute complex plays a central role, determining sensory organ mother cells and preventing neighbouring cells from following the same developmental pathway.