The question of which germ-line V kappa genes are expressed was studied by sequencing 70 different cDNA clones from a human spleen library and one clone from a fetal liver library. The sequences were compared to a data base containing all germ-line V kappa gene and pseudogene sequences. In addition, 51 rearranged genomic V kappa genes, 170 cDNA and 74 kappa proteins from the literature were assigned to specific germ-line V kappa genes and included in the comparisons. Not all the known, potentially functional V kappa genes were found to be expressed, while some genes with minor defects are. The total number of expressed genes is smaller than expected: so far 21 germ-line genes and 5 pairs of duplicated identical genes are known to be transcribed. The corresponding numbers for rearranged genomic V kappa genes and kappa proteins are 17 plus 4 and 7 plus 7, respectively. A second aim of the study was to find out whether the expressed repertoire contains derivatives of germ-line V kappa genes still missing in our data base; no evidence for the existence of such genes was found. Several cDNA clones contained additional nucleotides between the V kappa and J kappa gene segments, which may be germ-line derived, inserted by terminal deoxynucleotidyl transferase or introduced by other mechanisms. Somatic gene conversion seems not to play a major role in creating the human kappa gene diversity. Various aspects of the hypermutation of kappa genes are discussed and the formation of block mutations, i.e. the alterations of two or more adjacent nucleotides is stressed as a remarkable feature of the process.

Over the past decade, the spotted wing Drosophila, Drosophila suzukii, has invaded Europe and America and has become a major agricultural pest in these areas, thereby prompting intense research activities to better understand its biology. Two draft genome assemblies already exist for this species but contain pervasive assembly errors and are highly fragmented, which limits their values. Our purpose here was to improve the assembly of the D. suzukii genome and to annotate it in a way that facilitates comparisons with D. melanogaster. For this, we generated PacBio longread sequencing data and assembled a novel, high-quality D. suzukii genome assembly. it is one of the largest Drosophila genomes, notably because of the expansion of its repeatome. We found that despite 16 rounds of full-sib crossings the D. suzukii strain that we sequenced has maintained high levels of polymorphism in some regions of its genome. As a consequence, the quality of the assembly of these regions was reduced. We explored possible origins of this high residual diversity, including the presence of structural variants and a possible heterogeneous admixture pattern of North American and Asian ancestry. Overall, our assembly and annotation constitute a high-quality genomic resource that can be used for both high-throughput sequencing approaches, as well as manipulative genetic technologies to study D. suzukii. Drosophila suzukii (Matsumura, 1931), the spotted wing Drosophila (Diptera: Drosophilidae), is an invasive fruit fly species originating from eastern Asia that has spread since 2008 in major parts of America and Europe. This species is still expanding its distribution 1,2 and is classified as a major pest on a variety of berries and stone fruit crops 3. Its behavior and phenotypic traits are now the subject of intense scrutiny both in the lab and in the field (reviewed in 4). Understanding the biology and the population dynamics of D. suzukii benefits from the production and mining of genomic and transcriptomic data, as well as manipulative genetic technologies including functional transgenesis and genome editing 5-7. Yet, the efficacy of these approaches relies critically on high-quality genomic resources. Currently, two D. suzukii genome assemblies, obtained from two different strains, have been generated based on short-read sequencing technologies 8,9. The utility of these valuable genomic resources is limited by the

In continuation of our efforts to elucidate the immunoglobulin kappa locus of the mouse we analyzed 46 yeast artificial chromosomes (YACs) containing V kappa, J kappa and C kappa genes. The YACs, which were derived from DNA of C57BL/6 and C3H mice, ranged from 0.3-1.9 Mb in size. On the basis of hybridization with probes specific for the V kappa gene families a group of 13 YACs was selected for detailed analysis. The V kappa genes of the YACs were then characterized by hybridization to the family-specific probes and by the sizes of the EcoRI fragments on which they were found. This way evidence was obtained for 140 different V kappa gene signals on the YACs. Of these 63 had been characterized before on clones from a cosmid library of total mouse DNA (I. Zocher et al., Eur. J. Immunol. 1995. 25: 3326-3331) and 22 others were found now on cosmid clones derived from the YACs. Six V kappa genes of the previous study which were not found on the YACs are probably located outside of the kappa locus. The YACs were arrayed in a unique order establishing a YACs panel which most likely contains the whole kappa locus. The cosmid contigs and solitary cosmid clones which contain the 63 plus 22 V kappa gene signals mentioned above comprise about 2.0 Mb. Assuming that the remaining 55 V kappa genes are spaced at the same average distance of 24 kb, one may extrapolate to a locus size of 3.3 Mb.

Pigmentation is a diverse and ecologically relevant trait in insects. Pigment formation has been studied extensively at the genetic and biochemical levels. The temporality of pigment formation during animal development, however, is more elusive. Here, we examine this temporality, focusing on yellow, a gene involved in the formation of black melanin. We generated a protein-tagged yellow allele in the fruit fly Drosophila melanogaster, which allowed us to precisely describe Yellow expression pattern at the tissue and cellular levels throughout development. We found Yellow expressed in the pupal epidermis in patterns prefiguring black pigmentation. We also found Yellow expressed in a few central neurons from the second larval instar to adult stages, including a subset of neurons adjacent to the clock neurons marked by the gene Pdf. We then specifically examined the dynamics of Yellow expression domain and subcellular localization in relationship to pigment formation. In particular, we showed how a late step of re-internalization is regulated by the large low-density lipoprotein receptor-related protein Megalin. Finally we suggest a new function for Yellow in the establishment of sharp pigmentation pattern boundaries, whereby this protein may assume a structural role, anchoring pigment deposits or pigmentation enzymes in the cuticle.

The diversity of forms in multicellular organisms originates largely from the spatial redeployment of developmental genes [S. B. Carroll, Cell 134, 25–36 (2008)]. Several scenarios can explain the emergence of cis-regulatory elements that govern novel aspects of a gene expression pattern [M. Rebeiz, M. Tsiantis, Curr. Opin. Genet. Dev. 45, 115–123 (2017)]. One scenario, enhancer co-option, holds that a DNA sequence producing an ancestral regulatory activity also becomes the template for a new regulatory activity, sharing regulatory information. While enhancer co-option might fuel morphological diversification, it has rarely been documented [W. J. Glassford et al., Dev. Cell 34, 520–531 (2015)]. Moreover, if two regulatory activities are borne from the same sequence, their modularity, considered a defining feature of enhancers [J. Banerji, L. Olson, W. Schaffner, Cell 33, 729–740 (1983)], might be affected by pleiotropy. Sequence overlap may thereby play a determinant role in enhancer function and evolution. Here, we investigated this problem with two regulatory activities of the Drosophila gene yellow, the novel spot enhancer and the ancestral wing blade enhancer. We used precise and comprehensive quantification of each activity in Drosophila wings to systematically map their sequences along the locus. We show that the spot enhancer has co-opted the sequences of the wing blade enhancer. We also identified a pleiotropic site necessary for DNA accessibility of a shared regulatory region. While the evolutionary steps leading to the derived activity are still unknown, such pleiotropy suggests that enhancer accessibility could be one of the molecular mechanisms seeding evolutionary co-option.