The Cytogenetics of a Recessive Visible Mutant Associated with a Deficiency Adjacent to the Notch Locus in DROSOPHILA MELANOGASTER

The recessive visible faswb allele in Drosophila is an interband deletion between salivary band 3C5, 6 and 7. Heterozygosity for the deletion does not suppress recombination between faswb and mutant sites at Notch adjacent to it.--Df(1)w67k30, deficient for salivary bands 3C2 to 6, is the left of faswb. By crossing over within the homologous bit of interband retained in w67k30 and faswb, the two deficiencies can be linked. Cytologically, 3C7, "fused" to 3C5,6 in faswb, becomes "fused" to 3C1 when the two are coupled. In the double deletion, the recessive visible phenotype of the faswb "allele* is suppressed. Both w67k30 and faswb can be recovered by uncoupling the two deficiencies.--The data suggest that the mutant faswb does not represent a lesion at Notch; the entire gene or locus seems to be present. The interband deletion in faswb has secondarily moved an intact Notch locus to a foreign environment that interferes with its normal function. When faswb is linked to w67k30, the interference is eliminated and normal Notch functions resume.--The position of Notch on the salivary gland chromosome is reviewed in relation to the information obtained in these experiments.     

Cytogenetics of Notch Mutations Arising in the Unstable X Chromosome Uc of Drosophila melanogaster

A derivative of the unstable X chromosome, Uc, isolated in 1978 is still unstable and exhibits most of the genetic properties characteristic of the original Uc. This derivative, Df(1)cm-In, contains an inversion of the genes between bands 6F1-2 and 3D3-5 and a lethal deficiency between 6D5-7 and 6F1-2. This chromosome generated Notch mutations at a rate of 3.47 +/- 0.32% during seven consecutive generations. Cytological analysis of 50 Notch mutations of independent origin in the Df(1)cm-In chromosome showed that all of the 50 had an apparently identical deletion involving the region between 3D3-5 and 3C7-8 of the X chromosome. The results of in situ hybridization indicated that the extent of deletion in all of the 20 Notch deficiencies sampled from the 50 mentioned above involves about 10 kb of the sequences from the 3' end of the Notch locus. In addition to hypermutability and the accumulation of site-specific chromosome breaks, the Df(1)cm-In chromosome reinverts its inversion to the normal sequence and exhibits use of the existing chromosome breakpoints to generate new rearrangements.     

Deletion of an insulator element by the mutation facet-strawberry in Drosophila melanogaster

Eukaryotic chromosomes are thought to be subdivided into a series of structurally and functionally independent units. Critical to this hypothesis is the identification of insulator or boundary elements that delimit chromosomal domains. The properties of a Notch mutation, facet-strawberry (fa(swb)), suggest that this small deletion disrupts such a boundary element. fa(swb) is located in the interband separating polytene band 3C7, which contains Notch, from the distal band 3C6. The fa(swb) mutation alters the structural organization of the chromosome by deleting the interband and fusing 3C7 with 3C6. Genetic studies also suggest that fa(swb) compromises the functional autonomy of Notch by allowing the locus to become sensitive to chromosomal position effects emanating from distal sequences. In the studies reported here, we show that a DNA fragment spanning the fa(swb) region can insulate reporter transgenes against chromosomal position effects and can block enhancer-promoter interactions. Moreover, we find that insulating activity is dependent on sequences deleted in fa(swb). These results provide evidence that the element defined by the fa(swb) mutation corresponds to an insulator.     

Analysis of DNA interband regions 3A5/A6, 3C5-6/c7 and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes]

Using electron microscopic (EM) data on the formation of a novel band from the P-element material after its insertion in the interband and the procedure of P-target rescue, DNA interband regions 3A5/A6, and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes were cloned and sequenced. EM analysis of the 3C region have shown that the formation of the full-size 3C5-6/C7 interband requires a 880-bp DNA sequences removed by deletion Df(1)faswb. A comparison of DNA sequences of six bands, two of which were obtained in the present work and four were described earlier, demonstrated the uniqueness of each of them in the Drosophila genome and heterogeneity of their molecular organization. Interband 60E8-9/E10 contains gene rpl19 transcribed throughout the development, in particular in salivary glands. In the other interbands examined 5' and 3' nontranslated gene regions are located. These results suggest that Drosophila interbands may contain both housekeeping genes and regulatory sequences of currently inactive genes from adjacent bands.     

Overlapping deficiencies refine the map position of the sex-linked actin gene of Drosophila melanogaster

A ³H-labelled actin-specific probe was hybridized to Drosophila melanogaster X chromosomes heterozygous for deficiencies in the 5C region. The results suggest that the sex-linked actin gene resides in the overlap region of Df(1)C149 and Df(1)N73 at 5C3-4.     

Genetic background modifies inner ear and eye phenotypes of jag1 heterozygous mice

Mice heterozygous for missense mutations of the Notch ligand Jagged1 (Jag1) exhibit head-shaking behavior indicative of an inner ear vestibular defect. In contrast, mice heterozygous for a targeted deletion of the Jag1 gene (Jag1del1) do not demonstrate obvious head-shaking behavior. To determine whether the differences in inner ear phenotypes were due to the types of Jag1 mutations or to differences in genetic background, we crossed Jag1del1 heterozygous mice onto the same genetic background as the missense mutants. This analysis revealed that variation of the Jag1 mutant inner ear phenotype is caused by genetic background differences and is not due to the type of Jag1 mutation. Genome scans of N2 backcross mice identified a significant modifier locus on chromosome 7, as well as a suggestive locus on chromosome 14. We also analyzed modifiers of an eye defect in Jag1del1 heterozygous mice from this same cross.     

Intragenic Deletions and Salivary Band Relationships in Drosophila

In the absence of assumptions pertaining to the organization and function of chromomeric DNA, the cytogenetic analysis of intragenic deletions that start at Notch and spread to the right or left of the locus suggests that the recombinational gene is bilaterally associated with salivary band 3C7. Either there are two genes resolved as a single cistron, or one must seek an alternative interpretation that allows some modicum of independent in the relationship between gene and band. Although we momentarily lean toward the hypothesis that gene and salivary band are separate entities on a binemic chromosome, alternative views can be devised, and the data must remain open to reinterpretation.—The recessive visible allele fa^swb behaves as a point mutant at the left end of the map and seems to be a deletion in the interval 3C6 to 7; we suspect some part of the band is missing. We have used the aberration in fa^swb as a cytological marker, isolated intragenic recombinants, and subjected them to examination. The analysis indicates that the chromosomal interchanges occurred to the right of 3C7.     

The Role of Dosage of the Region 7d1–7d5-6 of the X Chromosome in the Production of Homeotic Transformations in DROSOPHILA MELANOGASTER

A high frequency of homeotic transformations appears in Df(3)red/+ progeny of Df(1)sn^C128 /+ females. Generally, the metathoracic appendages are partially transformed into mesothoracic ones. Df(1)sn^C128 includes a small region of the X chromosome: 7D1 to 7D5-6. Hypodosage of this region is mainly effective at the level of the maternal genotype, and the effect is probably due to hypodosage of the wild-type allele of the gene fs(1)h. Df(3)red has an effect that is mainly, if not exclusively, zygotic, probably due to hypodosage of the wild-type allele of Rg-bx. The frequencies of transformed flies resulting from the interaction between Df(1)sn^C128 and Df(3)red are not very sensitive to external conditions and genetic background. Studies of the interactions between Df(1)sn^C128 and other mutations or deficiencies of chromosome 3 [Rg-pbx, bx, pbx, Ubx¹, Ubx¹³⁰, Ubx⁸⁰, Df(3)P9] reveal an analogy between the hypodosage effect of region 7D1–7D5-6 and the effects of ether treatment of blastoderm stage eggs. The role of the gene fs(1)h in the process of segment determination is discussed in the light of these results.     

Redundant Notch1 and Notch2 Signaling Is Necessary for IFN�� Secretion by T Helper 1 Cells During Infection with Leishmania major

The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4⁺ T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4⁺ T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4⁺ T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2^ΔCD4Cre) were infected with the protozoan parasite Leishmania major. N1N2^ΔCD4Cre mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4⁺ T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4⁺ T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.     

Cytogenetic characterization of the 4BC region on the X chromosome of Drosophila melanogaster: Localization of the mei-9, norpA and omb genes

Thirty genetic alterations, which involve the 4BC region of the Drosophila X chromosome, have been induced by ionizing radiation or by an endogenous mutator element. These mutations were recovered by screening for reversion of the dominant mutants Oce and Qd or for induction of the recessive mutants bi and rb. Among the 23 mutants generated by ionizing radiation, 20 have proven to be cytologically detectable chromosomal aberrations. Seven additional unique aberrations were generated in the Uc mutator strain. In total, 22 cytologically detectable deficiencies, 3 translocations, 1 inversion, 1 transposition, and 3 cytologically normal mutants have been recovered. Complementation analysis has permitted the cytogenetic localization of eight genes in the 4BC region. The mei-9 locus has been assigned to region 4B4-6, because this function is carried by Df (1)rb ⁴¹ but not by Df(1)bi ^D1. The norpA locus has been placed in the 4B6-C1 region based on its location between the distal breakpoints of Df(1)bi ^D2 and Df(1)rb ⁴¹. The genes lac, Qd, bi, and omb are localized to bands 4C5,6, rb to 4C6 and amb to 4C7,8. With one exception the complementation analysis has also permitted a determination of the linear sequence of these genes. This cytogenetic localization of these loci will facilitate the cloning and molecular analysis of genes controlling a key function in DNA repair and recombination (mei-9), and two fundamental neural functions (norpA and omb).     

An extensive deletion causing overproduction of yeast iso-2-cytochrome c

CYC7-H3 is a cis-dominant regulatory mutation that causes a 20-fold overproduction of yeast iso-2-cytochrome c. The CYC7-H3 mutation is an approximately 5 kb deletion with one breakpoint located in the 5' noncoding region of the CYC7 gene, approximately 200 base from the ATG initiation codon. The deletion apparently fuses a new regulatory region to the structural portion of the CYC7 locus. The CYC7-H3 deletion encompasses the RAD23 locus, which controls UV sensitivity and the ANP1 locus, which controls osmotic sensitivity. The gene cluster CYC7-RAD23-ANP1 displays striking similarity to the gene cluster CYC1-OSM1-RAD7, which controls, respectively, iso-1-cytochrome c, osmotic sensitivity and UV sensitivity. We suggest that these gene clusters are related by an ancient transpositional event.     

The recombinational analysis of aberrations and the position of the notch locus on the polytene chromosome of Drosophila

Summary The recombinational analysis of heterozygotes for a point-mutant N and a deficiency N suggests that the map region approximated by the interval fa to nd ² is at the right edge of salivary band 3C7 or in the interband to the right. The map region N ^55ell to fa can be anywhere between the left interband and the right edge of 3C7. We discovered that small inversions also can be used in the recombinational analysis, and the inversion data support the conclusions already described.The reactivation of latent mutability in a Notch inversion resulted in reinversion of the original aberration, followed by reversion of N to N ⁺. From the same Notch inversion, we isolated a spontaneous deficiency superimposed upon the original aberration, which supported our hypothesis that two of our w to N deficiencies probably originated as deficiencies superimposed upon inversions.     

The nature of radiation-induced mutations at the white locus of Drosophila melanogaster

X-Ray- and neutron-induced mutations at the white locus of Drosophila melanogaster were used to study the nature of radiation-induced genetic damage. Genetic analysis showed the presence of multi-locus deficiencies in 15 out of 31 X-ray mutants and in 26 out of 35 mutants induced by neutrons. The DNA from 11 X-ray and 4 neutron mutants, which were not multi-locus deficiencies, was analyzed by Southern blot-hybridization. Deletions were observed in 2 X-ray and 1 neutron mutant. In combination with cytogenetic techniques, chromosomal rearrangements affecting the white locus (translocations, inversions, etc.) were identified in 3 X-ray and in 2 neutron mutants. A hot-spot for translocation breakpoints was identified in the left arm of the third chromosome. 5 X-ray mutants, which apparently did not contain large deletions, were subjected to further analysis by the nuclease S1 protection method, after cloning of the white gene. In 4 mutants a small deletion could indeed be detected in this way. Thus it seems that by far the main part of X-ray- and neutron-induced white mutants have arisen through large changes in the white gene, especially deletions.     

Characterization of four induced chromosome deficiencies in rice (Oryza sativa L.).

Four rice (Oryza sativa L.) deficiencies, involving chromosomes 4 (Df4), 8 (Df8), and 11 (Df11-1, Df11-2), were studied. The deficiencies were induced by means of the pseudodominance technique, i.e., strains carrying one or more recessive marker genes were fertilized with irradiated pollen of a strain carrying normal alleles at corresponding loci. No characteristic morphological features were found in the deficiencies, as compared with the normal F1 plants in the progeny. The deficiencies showed high or complete seed sterility. Genetic and cytological studies indicated deficiencies in chromosomes 4, 8, and 11. The fragment chromosomes in Df4, Df8, and Df11-2 were short, possibly being derived from the heterochromatin regions of the chromosomes, including kinetochores, and the fragment chromosome in Df11-1 was long, with about 75% of the long arm missing. At metaphase I, Df4, Df8, and Df11-2 showed only the chromosome configuration of 11 II (bivalents) + 2I (univalents), and Df11-1 only that of 12 II. It seems that the short fragments tend to stay as univalents in meiosis, probably because of their shortness. On the other hand, long fragments act as normal chromosomes and associate with their homologues. The deficiencies were not transmitted to the progenies, although only a few offspring were examined. By using the induced deficiencies Df4 and Df11-1, two morphological marker genes, lg (liguleless) and la (lazy growth habit), were located on the long arm of chromosomes 4 and 11, respectively. This is the first report in rice utilizing induced chromosome deficiencies to locate a gene on a specific arm of a chromosome. The use of induced deficiencies for studying the structure of the rice genome is discussed. Key words : rice, chromosome, deficiencies, cytology, transmission.     

Suppression of the facet-strawberry position effect in Drosophila by lesions adjacent to notch

The recessive visible rough eye mutant effect of fa ^swb, a small deletion at the 5' end of the Notch locus, is suppressed when fa^swb is coupled to five different closely linked deficiencies distal to salivary band 3C7. In addition, an inversion with a proximal breakpoint between 3C3 and 3C5 similarly suppresses the mutant effect. The data support the position effect interpretation of fa ^swb: The small deletion allows functions distal to Notch to interfere with functions at Notch, and when the interference is eliminated, the fa^swb-mutant effect disappears.—The fa^swb deletion also interacts with another recessive visible rough-eye mutant at Notch called fa^g. In the cis condition, fa^swb fa ^g double mutants have a mutant-eye phenotype like fa ^g (similar to the mutant effect of fa^swb) and, in addition, express an accessory phenotype (thickened wing veins). Although the mutant-eye effect of fa^swb can be suppressed by lesions adjacent to Notch, the accessory phenotype of the coupled mutants is not suppressed. It is suggested that the fa^swb deletion has two observable effects: One is a modifiable position effect causing the fa^swb rough-eye phenotype; the other is a stable effect exerted upon a 5-kb insertion that is the probable cause of the fa^g mutant expression, thus resulting in a wing effect that accompanies the eye effect of fa ^g.