Cytogenetic analysis of the 2B3-4-2B11 Region of the X chromosome of Drosophila melanogaster

Larvae homozygous or hemizygous for the l(l) t435 mutation located within the early ecdysteroid puff 2B5, or carrying a deletion of the 2B5 band, die at the end of the third larval instar. In the salivary gland chromosomes of these larvae only intermoult puffs are detected. If these salivary glands are incubated in vitro with 20-OH ecdysone for 6 h the intermoult puff 68 C remains large, some early puffs (74EF and 75B) are induced to 30–40% of their normal size, other early (63F) and all late puffs (62E, 78D, 82F and 63E) are not induced at all. Puff 2B5 reaches its normal size but does not regress after 6h incubation with 20-OH ecdysone, as it does in normal stocks. The data obtained in this study show the existence of a locus (or loci) in the band (puff) 2B5 which is necessary for the normal response of the salivary gland chromosomes to the hormone 20-OH ecdysone.     

Molecular cloning of the white locus region of Drosophila melanogaster using a large transposable element

We report the molecular cloning of a chromosome segment including the white locus of Drosophila melanogaster. This region was isolated using a deficiency extending from the previously cloned heat-shock puff sequences at 87A7 to a large transposable element containing the loci white and roughest.FB-NOF, a 7.5 kb element with partial homology to a family of inverted repeat sequences (Potter et al., 1980), is found very near the deficiency breakpoint, and is followed by DNA originating from the white locus region. Sequences totalling ˜60 kb surrounding this initial entry point were obtained by the cloning of successively overlapping fragments from a wild-type strain. Several rearrangement breakpoints have been mapped relative to the cloned DNA; these define the limits of the white locus and further differentiate the “white proximal region”, thought to function in gene regulation, from the remainder of the locus. Insertion of the dispersed repetitive element copia into the white locus is observed in strains carrying the white-apricot allele. Analysis of several white-apricot revertants suggests that copia insertion is responsible for the apricot eye color phenotype.     

Cytogenetical analysis of the 2B3-4-2B11 region of the X chromosome of Drosophila melanogaster

Summary Of 13 ecs mutations, which affect female fertility, as revealed by complementation analysis, 7 are chromosome rearrangements involving the br complementation group. The other six show no cytologically detectable rearrangements and behave as completely or partially noncomplementing ecs alleles. All viable combinations of these 13 mutations were characterized by partial or complete female sterility. Viable heterozygotes carrying any of these mutations and the rearrangements Df(1)sta, T(1,3)sta, Df(1)St490, previously localized distal to the ecs locus, were also sterile. Using deletions and an electrophoretic mobility variant from the Staket strain, a minor chorion gene S70 has been mapped. It had been thought this gene was located in the 2B3-5 region, and corresponded to the ecs locus. However, in the present study, this gene was shown to map in the region removed by Df(1)sta (1E1-2-2B3-4) but outside that removed by Df(1)At127 (1E1-2-2A1-2), i.e. within the 2A1-2-2B3-4 region which is distal to the ecs locus. Rearrangements and point mutations at the ecs locus that result in female sterility had no effect on synthesis of the chorion protein s70. It may therefore be suggested that the chorion protein gene is not functionally associated with the ecs locus and that sterility is caused not by disruptions of the chorion protein gene but by lesions in the ecs gene itself. Thus, an ecs product, which controlls cell sensitivity to ecdysterone is also necessary for female fertility. Data on the locations of lesions affecting female fertility indicate that at least two elements at the ecs locus are essential for this function: a cis-acting distal zone with no effect on viability and a sequence within the essential part of the ecs locus. A defect in either of these zones or their separation by chromosomal rearrangement leads to female sterility.     

Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes

Fragments from section 3 of the salivary gland X chromosome of D. melanogaster were dissected with a micromanipulator. The DNA was extracted, cut and ligated to a λ vector in a volume of a few nanoliters in an oil chamber monitored through a microscope. From about 10 pg of DNA we obtained 80 recombinant clones, a sample of which were analysed and shown to contain Drosophila DNA which hybridises in situ to the region of section 3 of the X chromosome. With this technique we can isolate clones from any desired region as small as 200 kb from the euchromatic arms of polytene chromosomes. This paper is dedicated to Professor W. Beermann on the occasion of his sixtieth birthday     

Microdissection of the fragile X region.

We have microdissected and cloned the region around the fragile site at Xq27.3 on the human X chromosome. All of the clones tested map to the Xq27-Xq28 region, and detailed mapping on a panel of somatic cell hybrids indicates that the microdissected library contains sequences derived from both sides of the fragile X mutation. Some of these clones give signals in rodent DNA. This library demonstrates the power of microdissection for the identification of potential coding sequences near a disease locus and provides a promising resource for the identification of the fragile X mutation.     

Cytogenetic analysis of the X-chromosome region 2B1-2-2B9-10 of Drosophila melanogaster

In salivary glands of yellow control stock the puffing pattern in the ecdysone-added artificial C46P medium was on the whole similar to that observed during larval development in vivo. However, underdevelopment of a series of late puffs and a delay in the regression of early puffs were observed. In addition a set of medium puffs not visible in vivo appeared. Late puffs differed from those developing in Grace medium.When salivary glands of homozygotes for the lethal dor ^lt187, a mutation that causes death in the third instar with no signs of ecdysone induction were incubated with ecdysterone, the development of puffs was restored, i.e., the puffing pattern of mutant cells in vitro practically did not differ from that in cells of the control stock. This implies that the dor ^lt187 lethal allele belongs to the class of ecdysone-deficient mutations.     

A highly polymorphic region in the X chromosome of Drosophila

Summary Many cloned regions of the Drosophila genome show minimal variation between strains in overall sequence arrangement. While restriction site polymorphisms occur and the location of transposable elements may vary from one strain to another, such changes appear to be relatively minor variations, superimposed on overall genome stability. In contrast to this general situation, we describe here a segment of the X chromosome that is highly polymorphic in four strains of D. melanogaster and in D. simulans. The strains differ in the presence and extent of a short duplication and the presence of repetitive DNA. These results suggest that different regions of the genome may be subject to different evolutionary constraints, with some regions being particularly prone to extensive changes, even within a single species.     

Cytogenetic analysis of region 2B3-4-2B11 of the X-chromosome of Drosophila melanogaster

About 160 kb of DNA were cloned from the 2B region of the X chromosome, where the early ecdysone puff develops and the ecs locus is located. On the physical map of this sequence the positions of 13 chromosome rearrangement breakpoints interfering with both puff development and the ecs locus proximally and distally, were plotted by means of in situ hybridization. The maximal size of the ecs locus is about 100 kb (between the breakpoint of In(1)Hw ^49c and the proximal end of Df(1)St472) The DNA sequences essential for normal puffing are located within the ecs locus between the In(1)br ^lt103 and Df(1)St472 breakpoints and comprise about 65 kb. Thus the puff develops as a result of ecs activation. Since Df(1)P154, which reduces the puff size and removes the proximal part of the ecs locus, does not prevent puff induction by ecdysone, while removing the distal part of the locus by Df(1)St469 completely stops development of the puff, we conclude that the regulatory zone of the locus, which reacts to hormone is located in the distal parts of both the puff and the locus, proximal to the breakpoint of In(1)br ^lt103 .Since In(1)br ^lt103 , Df(1)pn7b and Df(1)br ^R1 damage ecs but do not prevent puffing it is proposed that there is a second regulatory zone for this locus with a minimal size of 15–20 kb (between the breakpoints of Df(1)br ^R1 and In(1)br ^lt103). After cytogenetic and electron microscopic analysis of 2B puff formation it seems very likely that the site of puff formation is situated in the proximal part of 2B3-4 and after enhancement of ecs expression by hormone it spreads proximally to the 2B6 band which does not puff. When the puff regresses at puff stages (PS)10-11 its material does not condense completely and a zone of residual puffing joins the condensed material located distal to it. This material can give the impression of a separate band, designated 2B5 in Bridges' map. For convenience we propose to call the site giving rise to the puff as 2B3-5.     

Assignment of the gene for neuroendocrine protein 7B2 (SGNE1 locus) to mouse chromosome region 2[E3-F3] and to human chromosome region 15q11-q15

The gene for 7B2, a protein found in the secretory granules of neural and endocrine cells (gene symbol SGNE1) was localized to the E3-F3 region of mouse chromosome 2 and to the q11-q15 region of human chromosome 15. This was determined by in situ hybridization, using a mouse 7B2 cDNA and an intronic fragment of the corresponding human gene as probes. The respective locations of SGNE1 in the two species correlate with the conservation of loci between these subregions of mouse chromosome 2 and human chromosome 15. Clinically, the human SGNE1 DNA fragment may serve as a molecular probe of this locus in both the Prader-Willi and the Angelman syndromes, which are often accompanied by submicroscopic chromosomal deletions in the 15q11-15q13 region.     

Fine cytogenetical analysis of the band 10A1-2 and the adjoining regions in the Drosophila melanogaster X chromosome

The X chromosome region 9F12-10A7 (7 bands removed by Df(1)v ^l3) was saturated with lethal, semi-lethal, visible and male sterile mutations. A total of 11 complementation groups were found. In the more narrow interval of Df(1)v ^l1 which removes 3 bands (10A1-2, 10A3, 10A4-5) 6 loci were localised. — The band 10A1-2 consists of a sereis of 5 different subunits: (i) silent DNA where no functions were found — at the distal edge of the band; (ii) and (iii) two genes: v and 1(1)BP4; (iv) silent DNA in middle of the band, (v) locus sev on the proximal edge of the band. About 70% of the band's DNA was found to be silent. — Using the set of chromosome rearrangements removing different parts of the band it was shown that these five sequences may function independently from each other.     

Fine cytogenetical analysis of the band 10a1-2 and the adjoining regions in the Drosophila melanogaster X chromosome

The region 9E1-2 - 10B1-2 of the Drosophila melanogaster X chromosome was analysed under the light (LM) and the electron (EM) microscope using different fixatives and an EM map of the region was constructed. EM analysis revealed 21 bands in the region 9E1-2 - 10B1-2 instead of 36 bands in Bridges' map. This discrepancy mainly results from the fact that 14 bands indicated as "doublets" by Bridges appear as a single bands. No doublets were found in the whole 9B1-2 - 10C1-2 region after fixation of salivary glands in 3% glutaraldehyde, 3% formaldehyde and 3 : 1 ethanol-acetic acid mixture. 45% acetic acid is the only fixative which results in strongly vacuolated appearance of the bands. - The break points of 30 chromosome rearrangements in the region 9E1-2 - 10B1-2 were located under EM or LM within the limits of the EM map of this region.     

Molecular genetic variation in the centromeric region of the X chromosome in three Drosophila ananassae populations. II. The Om(1D) locus

Restriction-site and sequence-length polymorphism in the Om(1D) locus region on the X chromosome in Drosophila ananassae was investigated for three natural populations (from Burma, India, and Brazil), by using hexanucleotide-recognizing restriction enzymes. The estimates of average heterozygosity per nucleotide (pi) were 0.0085, 0.0043, and 0.0004 for the Burma, India, and Brazil populations, respectively, and the average frequencies of insertions/deletions were 0.078, 0.054, and 0.007/chromosome/kb. While the pi values at this locus are similar to the estimates obtained from other euchromatic loci in D. ananassae or in other Drosophila species, the frequencies of insertions/deletions are much higher than those previously reported from Drosophila. The exceptionally high frequencies of length polymorphisms in the Burmese sample and, to a lesser extent, in the Indian sample indicate that the hypermutability of Om(1D), caused by the frequent insertion of the transposable element tom, may be due to locus-specific rather than to tom element-specific properties. The low level of nucleotide variation in the Brazilian population seems to be due to a recent bottleneck of population size. This population was apparently founded in recent years by a small number of individuals and has been relatively isolated ever since.     

Microdissection and microcloning from the proximal region of mouse chromosome 7: isolation of clones genetically linked to the pudgy locus

Microdissection and microcloning have been utilized in order to create a bank of clones from the proximal region of mouse chromosome 7. Several important loci map to this area, including the albino locus (c), pink-eye dilution (p), and the developmental mutant, pudgy (pu). By use of interspecific crosses between Mus musculus domesticus and Mus spretus, we have generated backcross progeny segregating for the mutations chinchilla (cch) and pink-eye dilution (p). Exploiting the evolutionary divergence between the two species, we have analyzed the inheritance of restriction fragment length variants of three microclones and their linkage to the two markers cch and p, respectively. All three clones studied map to the dissected region, and as such also show genetic linkage to the pudgy locus. This bank of chromosome 7-derived microclones should provide molecular start points for the isolation of a variety of developmental loci of unknown gene product, including the pudgy locus.     

Heterochromatic ABO locus of the Drosophila melanogaster X chromosome overlaps with the region of localization of repeats, including mobile elements and Stellate genes

A loss of certain heterochromatic regions (ABO loci) of various chromosomes dramatically distorts the early embryo development in the progeny of females mutant for the abnormal oocyte (abo) gene, which is located in euchromatin of chromosome 2. One ABO locus (X-ABO) is in X-chromosomal heterochromatin distal of the nucleolus organizer. A cluster of the Stellate repeats is located in the same heterochromatin block. Deletions of various fragments from distal heterochromatin were tested for the effect on expression of the abo mutation. The X-ABO locus was assigned to X-chromosomal heterochromatin segment h26 and shown to include repeats consisting mostly of mobile elements and defective Stellate copies. A major part of the regular Stellate tandem repeats proved to be distal of the X-ABO locus.     

Chromosomal arrangement of heat shock locus 2-48B in Drosophila hydei

cDNA, copied from nuclear RNA isolated from heat shocked Drosophila hydei cells, has been cloned. From this collection of clones a clone, N09-15, with a 450 bp insert has been isolated that hybridizes in situ to the heat shock locus 2-48B of Drosophila hydei. The N09-15 sequence is present in two different genomic arrangements, as shown by restriction mapping, in our wild type D. hydei population. These genomic arrangements are allelic. Both alleles contain multiple copies of the N09-15 sequence but differ in their lengths and in the distribution of Msp I and Taq I sites.