Deltex, a Locus Interacting with the Neurogenic Genes, Notch, Delta and Mastermind in Drosophila Melanogaster

The Notch locus of Drosophila melanogaster, which codes for a transmembrane protein sharing homology with the mammalian epidermal growth factor, is one of a small number of zygotically acting genes, the so called neurogenic loci, which are necessary for the correct segregation of neural from epidermal lineages during embryogenesis. In an attempt to identify genes whose products may interact with that of Notch, we designed a genetic screen aimed at identifying suppressors of certain Notch mutations which are known to affect the extracellular epidermal growth factor homologous domain of Notch. Mutations in two neurogenic loci were identified as suppressors: Delta, whose product was recently shown to interact with Notch and mastermind. In addition, a third, X-linked gene was shown capable of acting as a suppressor. We show that this gene is the deltex locus, characterize the phenotype of deltex mutations, and demonstrate both a maternal and zygotic action of the locus. All deltex alleles behave as recessive viables affecting wing, ocellar and eye morphology. There are allele specific interactions between deltex and various Notch alleles; for example, deltex mutants with a reduced dosage of wild-type Notch die as pupae. deltex also interacts with Delta and mastermind in a fashion that is formally analogous to its interaction with Notch. These results emphasize the special relationship between Notch, Delta and mastermind suggested by previous work and indicate that deltex is likely to play an important role in the same genetic circuitry within which these three neurogenic loci operate.     

Is the Notch Locus of DROSOPHILA MELANOGASTER a Tandem Repeat? Correlation of the Genetic Map and Complementation Pattern of Selected Mutations

Correlation of the complementation relationships between the Notch locus alleles fa(g), fa(no), Ax(59d) and nd, with their genetic map order, suggests a tandem repetition within the locus of functionally related sites. This observation is discussed in relation to two hypotheses: (1) that the Notch locus contains a tandem repeat of genetic material; and (2) that the tertiary structure of the Notch locus product has a spiral configuration.     

Fs(1)yb Is Required for Ovary Follicle Cell Differentiation in Drosophila Melanogaster and Has Genetic Interactions with the Notch Group of Neurogenic Genes

Phenotypic and genetic analyses demonstrate that fs(1)Yb activity is required in the soma for the development of a subset of ovarian follicle cells and to support later stages of egg maturation. Mutations in fs(1)Yb cause a range of ovarian phenotypes, from the improper segregation of egg chambers to abnormal dorsal appendage formation. The mutant phenotypes associated with fs(1)Yb are very similar to the ovarian aberrations produced by temperature-sensitive alleles of Notch and Delta. Possible functional or regulatory interactions between fs(1)Yb and Notch are suggested by genetic studies. A duplication of the Notch locus partially suppresses the female-sterility caused by fs(1)Yb mutations, while reducing Notch dosage makes the fs(1)Yb mutant phenotype more severe. In addition, fs(1)Yb alleles also interact with genes that are known to act with or regulate Notch activity, including Delta, daughterless, and mastermind. However, differences between the mutant ovarian phenotype of fs(1)Yb and that of Notch or Delta indicate that the genes do not have completely overlapping functions in the ovary. We propose that fs(1)Yb acts as an ovary-specific factor that determines follicle cell fate.     

Modifications of the Notch Function by Abruptex Mutations in Drosophila Melanogaster

The function of the Notch gene is required in cell interactions defining alternative cell fates in several developmental processes. The Notch gene encodes a transmembrane protein with 36 epidermal growth factor (EGF)-like repeats in its extracellular domain. This protein functions as a receptor that interacts with other transmembrane proteins, such as Serrate and Delta, which also have EGF repeats in their extracellular domain. The Abruptex mutations of the Notch locus are associated with amino acid substitutions in the EGF repeats 24-29 of the Notch protein. We have studied, in genetic combinations, the modifications of Notch function caused by Abruptex mutations. These mutations lead to phenotypes which are opposite to those caused by Notch deletions. The Abruptex phenotypes are modified by the presence of mutations in other loci, in particular in the genes Serrate and Delta as well as Hairless, and groucho. The results suggest that all Abruptex mutations cause stronger than normal Notch activation by the Delta protein. Some Abruptex alleles also display an insufficiency of N function. Abruptex alleles which produce stronger enhancement of Notch activation also display stronger Notch insufficiency. This insufficiency could be due to reduced ability of Abruptex proteins to interact with Notch ligands and/or to form functional Notch dimers.     

Unequal crossingover between homologous chromosomes is not the major mechanism involved in the generation of new alleles at VNTR loci

To investigate the hypothesis that unequal exchange between homologous chromosomes is involved when new alleles are generated at VNTR loci, we used genetic linkage maps to identify flanking markers surrounding a VNTR marker locus. The minisatellite probe lambda MS1 was selected, as the hypervariable locus it detects undergoes spontaneous generation of new alleles in the germline at a rate of approximately 5%. Multipoint linkage analysis placed lambda MS1 within a cluster of polymorphic marker loci on chromosome 1p. Using the two closest flanking markers, CMM8 and YNZ2, we were able to characterize 12 new-allele events in terms of crossingover between the flanking markers. Statistical analysis of these data has allowed us to reject the model that assumes that events generating new alleles always involve unequal exchange between homologous chromosomes at meiosis.     

Recombination Can Initiate and Terminate at a Large Number of Sites within the Rosy Locus of Drosophila Melanogaster

This report presents the results of a recombination experiment designed to question the existence of special sites for the initiation or termination of a recombination heteroduplex within the region of the rosy locus. Intragenic recombination events were monitored between two physically separated rosy mutant alleles ry301 and ry2 utilizing DNA restriction site polymorphisms as genetic markers. Both ry301 and ry2 are known from previous studies to be associated with gene conversion frequencies an order of magnitude lower than single site mutations. The mutations are associated with large, well defined insertions located as internal sites within the locus in prior intragenic mapping studies. On the molecular map, they represent large insertions approximately 2.7 kb apart in the second and third exons, respectively, of the XDH coding region. The present study monitors intragenic recombination in a mutant heterozygous genotype in which DNA homology is disrupted by these large discontinuities, greater than the region of DNA homology and flanking both sides of the locus. If initiation/or termination requires separate sites at either end of the locus, then intragenic recombination within the rosy locus of the heterozygote should be eliminated. Contrary to expectation, significant recombination between these sites is seen.     

大麦基因组中的微卫星标记及其应用

微卫星是以少数几个核苷酸为单位多次串联重复的DNA序列,是一种简单序列重复(simple sequence repeats,SSR),两侧一般是保守序列。由于它具有多态性高、共显性、容易用PCR检测和结果稳定可靠等特点,因此是一种十分理想的分子标记。大麦的微卫星DNA随机分布于基因组中,平均每一个微卫星基因座有3～18个等位基因,最高可达37个。SSR标记已广泛用于分子遗传图谱的构建、遗传多样性研究、种质鉴定、主要性状基因的定位及分子标记辅助选择育种等。大多数SSR标记集中在着丝粒附近区域,1HL、5HL和6HS明显缺乏SSR标记。大麦的SSR标记还有待进一步的开发。 Microsatellite Markers and Applications in the Barley Genome FENG Zong-yun1,2,3,ZHANG Yi-zheng1,LING Hong-qing3 1.College of Life Sciences,Sichuan University,Chengdu 610065,China; 2.College of Agronomy,Sichuan Agricultural University,Ya'an 625014,China; 3.The State Key Laboratory of Plant Cell & Chromosome Engineering,Institute of Genetics & Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China Abstract:Microsatellites,also called simple sequence repeats (SSR),are simple,tandemly repeated DNA sequences with a repeat length of a few base pairs,and are very ideally used as molecular markers because of their abundance,high level of polymorphism,co-dominance and ease of assay with the polymerase chain reaction (PCR) by selecting primers as the conserved DNA sequences flanking the SSRs,as well as better stability.The experiments showed that SSRs are randomly distributed throughout the barley genome,and there are 3~18 alleles at a single SSR locus,up to 37 alleles/locus.SSR markers have being widely applied in the construction of molecular genetic map,the study of genetic diversity,the identification of germplasm,gene mapping for important traits and molecular marker-assisted selection.Meanwhile,most of markers are strongly clustered around the centromeric regions of all seven linkage groups.As a result of the clustering,genome coverage with SSRs remains incomplete with an obvious lack of markers on the long arms of chromosomes 1H and 5H and short arm of chromosome 6H.Therefore,it is very potential and necessary to further develop SSR markers in barley. Key words：barley;microsatellite marker;simple sequence repeats;genetic diversity;molecular mapping     

Allelic Negative Complementation at the Abruptex Locus of DROSOPHILA MELANOGASTER

The mutations of the Abruptex locus in Drosophila melanogaster fall into three categories. There are recessive lethal alleles and viable alleles. The latter can be divided into suppressors and nonsuppressors of Notch mutations. The recessive lethals are lethal in heterozygous combination with Notch. As a rule the recessive lethals are lethal also in heterozygous combination with the viable alleles. Heterozygous combinations of certain viable alleles are also lethal. In such heterozygotes, one heteroallele is a suppressor of Notch and the other is a nonsuppressor. Other heterozygous combinations of viable alleles are viable and have an Abruptex phenotype. The insertion of the wild allele of the Abruptex locus as an extra dose (carried by a duplication) into the chromosomal complement of the fly fully restores the viability of the otherwise lethal heterozygotes if two viable alleles are involved. The extra wild allele also restores the viability of heterozygotes in which a lethal and a suppressor allele are present. If, however, a lethal and a nonsuppressor are involved, the wild allele only partly restores the viability, and the effect of the wild allele is weakest if two lethal alleles are involved. It seems likely that of the viable alleles the suppressors of Notch are hypermorphic and the nonsuppressors are hypomorphic. The lethal alleles share properties of both types, and are possibly antimorphic mutations. It is suggested that the locus is responsible for a single function which, however, consists of two components. The hypermorphic mutations are defects of the one component and the hypomorphic mutations of the other. In heterozygotes their cumulative action leads to decreased viability. The lethal alleles are supposed to be defects of the function as a whole. The function controlled by the locus might be a regulative function.     

Genetic Organization of the agouti Region of the Mouse

The agouti locus on mouse chromosome 2 acts via the hair follicle to control the melanic type and distribution of hair pigments. The diverse phenotypes associated with various agouti mutations have led to speculation about the organization of the agouti locus. Earlier studies indicated that two presumed agouti alleles, lethal yellow (Ay) and lethal light-bellied nonagouti (ax), are pseudoallelic. We present genetic data showing probable recombination between Ay and three agouti mutations (at, a, and ax), which suggest that Ay is a pseudoallele of the agouti locus. The close linkage of an endogenous ecotropic murine leukemia provirus, Emv-15, to Ay provides a molecular access to genes at or near the agouti locus. However, previous studies suggested that the Emv-15 locus can recombine with some agouti alleles and therefore we analyzed mice from recombinant inbred strains and backcrosses to measure the genetic distance between various agouti alleles and the Emv-15 locus. Our data indicate that the Emv-15 locus is less than 0.3 cM from the agouti locus. These experiments provide a conceptual framework for initiating chromosome walking experiments designed to retrieve sequences from the agouti locus and give new insight into the genetic organization of the agouti region.     

Making sense out of missense mutations: Mechanistic dissection of Notch receptors through structure-function studies in Drosophila

Notch signaling is involved in the development of almost all organ systems and is required post-developmentally to modulate tissue homeostasis. Rare variants in Notch signaling pathway genes are found in patients with rare Mendelian disorders, while unique or recurrent somatic mutations in a similar set of genes are identified in cancer. The human genome contains four genes that encode Notch receptors, NOTCH1-4, all of which are linked to genetic diseases and cancer. Although some mutations have been classified as clear loss- or gain-of-function alleles based on cellular or rodent based assay systems, the functional consequence of many variants/mutations in human Notch receptors remain unknown. In this review, I will first provide an overview of the domain structure of Notch receptors and discuss how each module is known to regulate Notch signaling activity in vivo using the Drosophila Notch receptor as an example. Next, I will introduce some interesting mutant alleles that have been isolated in the fly Notch gene over the past > 100 years of research and discuss how studies of these mutations have facilitated the understanding of Notch biology. By identifying unique alleles of the fly Notch gene through forward genetic screens, mapping their molecular lesions and characterizing their phenotypes in depth, one can begin to unravel new mechanistic insights into how different domains of Notch fine-tune signaling output. Such information can be useful in deciphering the functional consequences of rare variants/mutations in human Notch receptors, which in turn can influence disease management and therapy.     

Both synchronous and asynchronous muscle isoforms of projectin (the Drosophila bent locus product) contain functional kinase domains

In Drosophila, the large muscle protein, projectin, has very different localizations in synchronous and asynchronous muscles, suggesting that projectin has different functions in different muscle types. The multiple projectin isoforms are encoded by a single gene; however they differ significantly in size (as detected by gel mobility) and show differences in some peptide fragments, presumably indicating alternative splicing or termination. We now report additional sequence of the projectin gene, showing a kinase domain and flanking regions highly similar to equivalent regions of twitchin, including a possible autoinhibitory region. In spite of apparent differences in function, all isoforms of projectin have the kinase domain and all are capable of autophosphorylation in vitro. The projectin gene is in polytene region 102C/D where the bentD phenotype maps. The recessive lethality of bentD is associated with a breakpoint that removes sequence of the projectin kinase domain. We find that different alleles of the highly mutable recessive lethal complementation group, l(4)2, also have defects in different parts of the projectin sequence, both NH2-terminal and COOH- terminal to the bentD breakpoint. These alleles are therefore renamed as alleles of the bent locus. Adults heterozygous for projectin mutations show little, if any, effect of one defective gene copy, but homozygosity for any of the defects is lethal. The times of death can vary with allele. Some alleles kill the embryos, others are larval lethal. These molecular studies begin to explain why genetic studies suggested that l(4)2 was a complex (or pseudoallelic) locus.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: 3. Frequent occurrence of genetic variants in some abundant polypeptides of PHA-stimulated peripheral blood lymphocytes

Summary The 100 or so most intensely Coomassie blue-stained polypeptides from PHA-stimulated peripheral blood lymphocytes were analyzed by two-dimensional electrophoresis in combination with family and population studies. Besides polymorphic lymphocyte cytosol 64k polypeptide reported previously, genetic variants were frequently observed in three polypeptides with molecular weights of 100,000, 49,000, and 40,000. All of them occur in the cytosol. These variant polypeptides are charge variants, because they are separated in the isoelectric focusing dimension. It is indicated by family and population studies and cell distribution analysis that the polypeptide with a molecular weight of 100,000 shows a genetic polymorphism determined by two alleles at a new autosomal locus, as described in the following paper. Family and population studies also suggest that a genetic polymorphism defined by alleles at an autosomal locus is present in each of the polypeptides with molecular weights of 49,000 and 40,000. In contrast to the previous reports of the extremely restricted genetic variability of the 100 or so most abundant fibroblast polypeptides, the present data indicate that common genetic variants are present at least in four of the 100 or so most intensely Coomassie blue-stained lymphocyte polypeptides. The result also shows that careful side-by-side comparison of two-dimensional electrophoresis patterns among both parents and their children is an effective method to detect genetic variant polypeptides.     

Comparative sequencing of a microsatellite locus reveals size homoplasy within and between european oak species (<Emphasis Type="Italic">Quercus</Emphasis> spp.)

Microsatellites (simple sequence repeats [SSRs]) are highly variable molecular markers that are a rich and readily assayed source of variation for population genetic studies. Cross-amplification between closely related species is possible when there are no (or few) sequence differences in the primer binding sites. The occurrence of nonhomologous fragments of the same size (size homoplasy) is a contraint of microsatellites. Size homoplasy can be caused by insertions/deletions (indels) in SSR flanking regions. We found that size variation in locus ssrQZAG9 is due to different repeat numbers of the SSR motifs but also to indels in SSR flanking regions. Indels were found within species belonging to sectionsRobur andCerris of genusQuercus and also between species of the 2 sections. In sectionRobur (Quercis robur L.,Quercus petraea [Matt.] Liebl.,Quercus pubescens Willd.), we detected rare alleles with an indel of 57 bp or 62 bp followed by a smaller indel of 12 bp in the SSR flanking regions. These alleles show a size range overlapping with that of alleles amplified inQuercus cerris L. (sectionCerris). Multiple alignments with sequences of sectionRobur revealed the same SSR repeat motif but multiple indels in SSR flanking regions inQ. cerris. We discuss the effects of size homoplasy of SSR loci for the study of interspecific gene flow and on estimates of population differentiation.     

Genetic localization and sequential electrophoresis of glucose-6-phosphate dehydrogenase in Drosophila melanogaster

Studies were undertaken to investigate two critical aspects of the glucose-6-phosphate dehydrogenase polymorphism in Drosophila melanogaster. The first investigation unequivocally maps the genetic site of the G6PD locus to the X chromosome. The second study subjects a set of isochromosomal lines to sequential electrophoresis in an attempt to uncover common molecular heterogeneity within the global polymorphism, assuming that this variation may have gone undetected under conventional electrophoretic conditions. The genetic site was mapped following the segregation of the two common electrophoretic alleles, a so-called null allele, and two rare electrophoretic variants. From the pooled results, the Zw locus mapped to 62.9 on the X chromosome relative to the flanking markers car (at 62.5) and sw (at 64.7). A set of 126 iso-X chromosomal lines of diverse geographic origin was subjected to sequential electrophoresis under three different acrylamide conditions in addition to the conventional starch electrophoretic system. No additional variation beyond the common diallele polymorphism was seen.     

Notchless encodes a novel WD40-repeat-containing protein that modulates Notch signaling activity.

Signaling by Notch family receptors is involved in many cell-fate decisions during development. Several modifiers of Notch activity have been identified, suggesting that regulation of Notch signaling is complex. In a genetic screen for modifiers of Notch activity, we identified a gene encoding a novel WD40-repeat protein. The gene is called Notchless, because loss-of-function mutant alleles dominantly suppress the wing notching caused by certain Notch alleles. Reducing Notchless activity increases Notch activity. Overexpression of Notchless in Xenopus or Drosophila appears to have a dominant-negative effect in that it also increases Notch activity. Biochemical studies show that Notchless binds to the cytoplasmic domain of Notch, suggesting that it serves as a direct regulator of Notch signaling activity.     

Temperature-Sensitive Mutations of the Notch Locus in DROSOPHILA MELANOGASTER

Temperature-conditional mutations of the Notch locus were characterized in an attempt to understand the organization of a "complex locus" and the control of its function in development. Among 21 newly induced Notch alleles, about one-half are temperature-conditional for some effects, and three are temperature-sensitive for viability. One temperature-sensitive lethal, l(1)N^ts1, is functionally non-complementing for all known effects of Notch locus mutations and maps at a single site within the locus. Among the existing alleles involved in complex patterns of interallelic complementation, Ax^59d5 is found to be temperature-sensitive, while fa ^g, spl, and l(1)N are temperature-independent. Whereas temperature-sensitive alleles map predominantly to the right-most fifth of the locus, fa^g, spl, and l(1)N are known to map to the left of this region. Temperature-shift experiments demonstrate that fa^g, spl, and l(1)N cause defects at specific, non-overlapping times in development.—We conclude (1) that the Notch locus is a single cistron (responsible for a single functional molecule, presumably a polypeptide); (2) that the right-most fifth of the locus is, at least in part, the region involved in coding for the Notch product; (3) that the complexity of interallelic complementation is a developmental effect of mutations that cause defects at selected times and spaces, and that complementation occurs because the mutant defects are temporally and spatially non-overlapping; and (4) that mutants express selected defects due to critical temporal and spatial differences in the chemical conditions controlling the synthesis or function of the Notch product. The complexity of the locus appears to reside in controlling the expression (synthesis or function) of the Notch product in development.     

The Study of Genic Variation by Electrophoretic and Heat Denaturation Techniques at the Octanol Dehydrogenase Locus in Members of the DROSOPHILA VIRILIS Group

Heat denaturation studies of three different electrophoretic allozymes of octanol dehydrogenase were performed from 10 species of the Drosophila virilis group. A total of 18 alleles were discovered, and in 3 species in which electrophoretic studies indicated that the locus was monomorphic, heat denaturation showed that the locus was polymorphic. We show that electrophoretic studies underestimate the number of alleles at this locus by a factor of 2.6 in these species. The results are discussed in the light of the continuing controversy over selection and neutral theories of genetic variation.     

Molecular Markers for the agouti Coat Color Locus of the Mouse

The agouti (a) coat color locus of the mouse acts within the microenvironment of the hair follicle to control the relative amount and distribution of yellow and black pigment in the coat hairs. Over 18 different mutations with complex dominance relationships have been described at this locus. The lethal yellow (Ay) mutation is the top dominant of this series and is uniquely associated with an endogenous provirus, Emv-15, in three highly inbred strains. However, we report here that it is unlikely that the provirus itself causes the Ay-associated alteration in coat color, since one strain of mice (YBR-Ay/a) lacks the provirus but still retains a yellow coat color. Using single-copy mouse DNA sequences from the regions flanking Emv-15 we have detected three patterns of restriction fragment length polymorphisms (RFLPs) within this region that can be used as molecular markers for different agouti locus alleles: a wild-type agouti (A) pattern, a pattern which generally cosegregates with the nonagouti (a) mutation, and a pattern which is specific to Emv-15. We have used these RFLPs and a panel of 28 recombinant inbred mouse strains to determine the genetic linkage of these sequences with the agouti locus and have found complete concordance between the two (95% confidence limit of 0.00 to 3.79 centimorgans). We have also physically mapped these sequences by in situ hybridization to band H1 of chromosome 2, thus directly confirming previous assignments of the location of the agouti locus.