Notch Locus Research Articles

Induction of notch-like phenocopies by methoxyacetate dependent on alcohol dehydrogenase allozymes of Drosophila melanogaster

1. 1. Third instar larvae of two wild type strains of Drosophila melanogaster, homozygous for Adh 71 k and Adh F respectively were exposed to methoxyacetate, an inhibitor of sarcosine dehydrogenase. The two strains showed a different Notch-like phenocopy frequency. This is explained as a consequence of the different oxidation rates of sarcosine by the two alcohol dehydrogenase allozymes, ADH 71k and ADH F. 2. 2. Inhibition of ADH activity in vivo by acetone, before the administration of methoxyacetate, enhanced in both strains differentially the frequency of wing-notches and mortality. Then the phenocopy frequency in the Adh 71 k strain almost equalled that of the Adh F -strain. 3. 3. The activity of sarcosine dehydrogenase is controlled by the Notch locus. Activity of sarcosine dehydrogenase is lowered by Notch mutations and methoxyacetate, whereas at the same time they do not affect the activity of alcohol dehydrogenase. 4. 4. Therefore we suppose that ADH 71k forms a bypass for sarcosine oxidation, when in vivo sarcosine dehydrogenase activity is reduced either by artificial in vivo inhibition or by a mutation. 5. 5. This explains also the fixation of the Adh 71 k allele in stocks with Notch mutants of Drosophila melanogaster.

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).

The molecular genetics of the Notch locus in Drosophila melanogaster

The Notch locus of Drosophila melanogaster profoundly affects differentiation of the central nervous system in the early embryo. Previous molecular studies suggested that the locus spans 40 kb of DNA and encodes a 10.5-kb poly(A) + RNA. The results of genetic, cytogenetic, and molecular studies of newly induced and preexisting Notch alleles are reported. Molecular analysis of 5′ flanking mutations defines a distal limit for the locus, and transcriptional activity of Notch in relation to that of flanking transcription units provides evidence regarding the proximal limit. Examination of a set of mutable alleles implicates a foldback transposable element as the basis of chromosomal instability, and shows that insertion sequences within the locus do not necessarily result in a mutant phenotype. The results are discussed with regard to existing developmental and genetic analyses, and a molecular model is proposed that attempts to explain the pleiotropic action of Notch.

Opa: A novel family of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. melanogaster

The principal transcription product of Notch, a locus involved in the neurogenesis of D. melanogaster, is a developmentally regulated poly(A) + RNA approximately 10.5 kb in length. Analysis of the structure of this RNA has revealed a 93 bp repeated sequence that is shared by many other developmentally regulated transcription units. Nucleotide sequence analysis of the repeat shows an unusual structure consisting predominantly of the triplets CAG and CAA, both of which can code for the amino acid Gin. We present evidence indicating that the Notch repeat is a member of a novel family of repetitive elements, which we term the opa family. Our data suggest that some of these elements may be not only transcribed but also translated. We compare opa with other known transcribed repeats and speculate on its functional significance.

Evidence for a multiple function of the alcohol dehydrogenase allozyme ADH71k of Drosophila melanogaster

Alcohol dehydrogenase of Drosophila melanogaster catalyzes the oxidation of many primary and secondary alcohols. We show that sarcosine, choline and dihydroorotate are substrates of ADH in vitro. The first two substrates are regular substrates of the choline shunt, and the latter of the de novo pyrimidine synthesis. Differences in oxidative ability towards sarcosine and dihydroorotate between two ADH allozymes, ADH71k and ADHF, are observed. The catalytic activity of ADH71k towards sarcosine and dihydroorotate might be responsible for its allelic fixation in Notch8 mutant stocks, in which Notch females have a decreased level of the regular enzymes for these substrates. Their oxidation by ADH71k might act as a bypass, which restores at least part of the decreased activity of enzymes encoded by the Notch locus.

The Notch locus of Drosophila melanogaster

The Notch locus appears to correspond to a 37 kb transcription unit. Homologous poly(A) + RNA is about 11.7 kb. Nine RNA coding regions have been detected and localized within the transcription unit by S1 nuclease analyses. These range in size from 130 to 7250 bp. Sequences within the 3′ coding region are variably used. Twenty-four Notch locus “point” mutations have been examined. Seven are associated with DNA insertions. Four insertions are associated with dominant mutations and are located within or very near RNA coding sequences of the 37 kb transcription unit. Three insertions are associated with recessive mutations and fall within intervening sequences. The positions of all insertions agree with the genetic map. It is estimated that, in the Notch region, one map unit corresponds to approximately 275 kb. On this basis, most of the 17 mutations that were not associated with DNA insertions can be placed within coding sequences of the 37 kb transcription unit.

Molecular cloning of Notch, a locus affecting neurogenesis in Drosophila melanogaster.

The Notch locus is one of the best characterized loci in Drosophila melanogaster in terms of its genetic structure and developmental effects. Mutations in this locus profoundly affect the differentiation of the early embryo. Using an inversion involving the Notch locus and previously cloned sequences, we have isolated chromosomal segments from the Notch region (3C7) encompassing 80 kilobases (kb) of DNA. Based on comparison between mutant and wild-type DNA, we have positioned cloned sequences within the Notch genetic map; furthermore, we have defined a region of approximately 40 kb within which the structural lesions correlating with all Notch alleles mapped to date appear to reside. We have examined the transcriptional activity of the cloned sequences during ontogeny and find a single size class of poly(A)+ RNA, 10.5 kb long, that is homologous to sequences within this 40-kb region. We conclude that DNA sequences belonging to the Notch locus have been cloned and that the 10.5-kb poly(A)+ RNA is essential for wild-type Notch function. We discuss these structural and transcriptional data in light of the existing genetic and developmental characterization of the Notch locus.

THE NOTCH LOCUS OF DROSOPHILA HYDEI: ALLELES, PHENOTYPES AND FUNCTIONAL ORGANIZATION

The Notch (N) locus of Drosophila hydei and a series of its alleles and phenotypes are described. Some models are discussed to explain the opposite effects of some alleles on the structure of the wing, the neomorphic action of N(Ax) over typical N alleles and the interaction with the mutation Costal-nick (Cnk).

The Notch locus of Drosophila melanogaster: A molecular analysis

AbstractGenetic analysis has suggested that neurogenesis in D melanogaster is under the control of a small number of genes. We have initiated a molecular study of the genes involved in this developmental event and started our analysis with the Notch locus, which is one of the best characterized loci in D melanogaster in terms of its genetic structure and developmental effects. In this paper we report on the molecular characterization of the Notch locus.We describe the molecular cloning of Notch and present evidence that the entire locus is defined by approximately 40 kb of genomic DNA. The transcriptional activity of these sequences during development has been examined and the results indicate that an approximately 10.5‐kb‐long poly A+ RNA is essential for wild type Notch activity. Mapping of this RNA within the physical map of Notch indicates that it is the processed product of an approximately 40‐kb primary transcription unit spanning the entire Notch locus. More detailed analysis of the 10.5 kb RNA localizes several exons and identifies a small repetitive sequence that seems to be present in the mature Notch transcript. Structural details of a selected number of Notch locus mutations are presented and discussed. Preliminary data on the molecular structure of Notch‐homologous DNA sequences in closely related species are also presented.

The action of the Notch locus in Drosophila melanogaster III. Biochemical effects of recessive visible mutations on mitochondrial enzymes

The effects of six recessive visible Notch mutations on the activities of four enzymes of the mitochondrial respiratory chain are described. Their effects in hemizygous condition in males are similar to those of the recessive lethal Notch mutations in heterozygous condition. This explains their viability. The characteristic morphological Notch expression cannot be related to the different activity patterns of four enzymes caused by the recessive visible mutations. Whereas there is a correspondence between the location of the recessive lethals and the recessive visibles in relation to their enzyme activity patterns, this is not so in relation to their morphological effects. In contrast to the enzyme activity determinations in heterozygotes for recessive lethals, the effects of recessive visibles are determined in the hemizygous condition, thereby excluding the influence of wildtype Notch alleles. Such an influence is found in heterozygotes for the exceptional fanomutant, in which morphological expression is dependent on the wildtype X chromosome.

The action of the notchlocus in Drosophila melanogaster. II. Biochemical effects of recessive lethals on mitochondrial enzymes.

We show that six mapped recessive lethal point mutations of the Notch locus affect mitochondrial enzyme activities: NADH oxidase, NADH dehydrogenase, succinate dehydrogenase and alpha-glycerophosphate dehydrogenase. The mutant N264-40, which has the same morphological and embryological effects as the Notch8 deletion, demonstrates the same biochemical effects and dosage relations as Notch8. The other five mapped recessive lethals also affect four enzymic activities. They show specific patterns of activity that depend in several cases on the wild-type chromosome in the heterozygous females. That effect occurs with mutants located in the extreme right part of the Notch locus where some mutations, according to other authors, show temperature-sensitive expression.

The action of the notch locus in Drosophila melanogaster. I. Effects of the notch8 deficiency on mitochondrial enzymes.

It is shown that the Notch8 deficiency in Drosophila melanogaster affects a number of enzyme activities localized in the mitochondria, such as NADH oxidase (activity of the complete respiratory chain), NADH dehydrogenase (the first step in the respiratory chain before transfer to ubiquinone), Succinate dehydrogenase and alpha-glycerophosphate dehydrogenase. The experiments reported here do not exclude the possibility of involvement of other genes in the deficiency. The effect of duplications of the Notch locus on NADH oxidase and NADH dehydrogenase suggest that the locus determines the enzyme activities. The dosage effects of the Notch locus on activity suggest that this locus contains the structural genes for these enzymes.

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

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 nd2 is at the right edge of salivary band 3C7 or in the interband to the right. The map region N55ell 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.

Mutations of early neurogenesis inDrosophila.

Embryonic lethal mutations at the Notch locus are known to produce a conspicuous central nervous system hypertrophy accompanied by a hypotrophy of the epidermal sheath. We have studied several zygotic mutants belonging to four different autosomal complementation groups which produce the same phenotype. The embryonic development of the new mutants, as well as that of Notch, consists of an initial enlargement of the neurogenic region at the expenses of epidermal cell precursors. The possibility is discussed that these five loci are involved in the determination of neural and epidermal cell precursors.

Aminopyridines mimic mutant Drosophila developmental defects

1. The potassium blocking agents 2- and 3-aminopyridine cause developmental defects in Drosophila melanogaster when these agents are fed to third-instar larvae. The developmental defects thus produced are similar in appearance to the developmental phenotypes produced by mutations at the shibire and Notch loci. 2. Aminopyridine drugs enhance neuromuscular transmission at the Drosophila larval neuromuscular junction. The effect can be attributed to their potassium-blocking action in the presynaptic nerve terminal.

Cell culture of individual Drosophila embryos: II. Culture of X-linked embryonic lethals

ABSTRACT Results are reported from the culturing in vitro of cells from individual early gastrulae of the following four groups of X-linked embryonic lethal mutants of Drosophila melanogaster. (1) Notch lethals. Five Notch mutants were studied which have been reported to give similar abnormalities in whole embryos: the nervous system displays a three-fold hypertrophy as part of a shift in the pattern of differentiation within ectodermal derivatives, and mesodermal derivatives do not differentiate. An hypertrophy of nerve was found in cell cultures prepared from embryos of all five mutants. In addition, four of the five alleles consistently gave abnormalities of muscle differentiation: when compared to controls, Notch cultures had a reduced frequency of myotubes, and displayed unusual clusters of myocytes which had either failed to fuse or had fused incompletely. Results from mixed cultures prepared from two embryos were consistent with the autonomous expression of nerve and muscle abnormalities by Notch-8 cells in the presence of wild-type cells. It is argued that the Notch locus has a direct role in the differentiation of both nerve and muscle. (2) white deficiencies. Cells carrying either of two deficiencies gave a clear-cut pattern of abnormalities: initial cellular differentiations were normal, but nerve, muscle and fat-body cells progressively deteriorated during the culture period. Mixed cultures showed that wild-type cells could not ‘rescue’ mutant muscle and fat-body cells; however, the status of the autonomy of mutant nerve abnormalities in these cultures was unclear. Both white deficiencies remove cytological band 3C1, and this permits a comparison of results with those from cultures of cells from Notch-S embryos (also deficient for 3C1). Abnormalities displayed in cultures of the two types of mutant show no overlap. Therefore no consistent cellular abnormality can be attributed to absence of band 3C1. (3) lethal(l)myospheroid. In contrast to earlier observations on in vitro cell cultures (Donady & Seecof, 1972) muscle was seen to differentiate, though its morphology was extremely abnormal. Observations indicated that all cell types within the cultures had poor properties of adhesion to a glass substrate. It is argued that the observed abnormalities are not consistent with a mutant lesion which is restricted to the basement membrane (contraWright, 1960), and that all cell types carry a basic defect which may reside in the cell membrane. (4) shibirets alleles. Cultures of two temperature-sensitive lethal shibire alleles (shils1, shits3) were normal at the permissive temperature of 22 °C. At the restrictive temperature (29° C) early cell differentiation was normal but subsequent development was blocked. This blockage could be partially reversed by shifting cultures to the permissive temperature after as much as 10 days exposure to the high temperature. It is suggested that shits cells are mutant in a process which is basic to several cell types.

Temperature-sensitive periods and autonomy of pleiotropic effects of l(1) Nts1 , a conditional notch lethal in Drosophila

Analysis of temperature-shift experiments using strains homo- and/or hemizygous for a temperature-sensitive (ts) mutation of the Notch locus, l(1) N ts1 , has permitted us to localize temperature-sensitive periods (TSPs) both for lethality and for adult ectodermal morphology defects. Discrete TSPs for lethality are localized to the first half of the embryonic period, to the second larval instar, to the third larval instar, and to a 15 hr period immediately after pupation. TSPs for adult morphology defects are localized to the second and third larval instars for eyeless-headless and duplicated antenna, to the third larval instar for small and rough ( spl-like) eye, eye scar, fused leg segments, shortened tarsal leg segments, Notch wings, and extra macrochaetae, and to the early pupal period for extra and missing microchaetae, fa g -like rough eye and thick wing vein defects. Within the third larval instar, distinct patterns of eye, wing, and leg defects are observed. There is a striking similarity between the adult morphology defects and TSPs of l(1) N ts1 and those of the larval and adult locomotor mutant, shi ts1 ( C. A. Poodry, L. Hall, and D. T. Suzuki, 1973, Develop. Biol. 32, 373–386 ). Expression of l(1) N ts1 also has been studied in genetic mosaics, in which we find that the pleiotropic effects of l(1) N ts1 are autonomously expressed.

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.

Negative complementation at the notch locus of Drosophila melanogaster.

Four Abruptex alleles (AxE1, AxE2, Ax9B2, and Ax16172) have been mapped within the Notch locus. Based on their visible phenotypes and their interactions with one another and with N mutations, the Ax alleles can be divided into two groups. Heterozygous combinations of members of the same group are intermediate in phenotype compared to the respective homozygotes, whereas heterozygotes of Ax alleles from different groups exhibit negative heterosis, being much less viable and more extremely mutant than either homozygote. It is suggested that the Notch locus is a multi-functional regulator ("integrator") gene, whose product possesses both "repressor" and "activator" functions for the processes it regulates.

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)Nts1, 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, Ax59d5 is found to be temperature-sensitive, while fag, spl, and l(1)N are temperature-independent. Whereas temperature-sensitive alleles map predominantly to the right-most fifth of the locus, fag, spl, and l(1)N are known to map to the left of this region. Temperature-shift experiments demonstrate that fag, 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.