Electron microscopical analysis of Drosophila polytene chromosomes

An electron microscopic (EM) analysis was performed on regions of Drosophila melanogaster polytene chromosomes that contain inserted DNA segments of 19 and 8 kb. These segments had been inserted by P-elementmediated transformation. The 19 kb segment includes both the Drosophila hsp70 gene fused to the Escherichia coli -galactosidase gene and the rosy gene (Lis et al. 1983). This insert generates a new moderate-size band at the 9D4-9E1-2 region in polytene chromosomes. Upon heat shock, a puff originates from a portion of the new band. The 8 kb segment includes the Sgs7 and Sgs3 genes (Richards et al. 1983). This insert generates very diffuse thin bands that decondense at the stage of activation of the Sgs genes to produce wide interbands or small puffs. In all of the above cases, the insertion appears to occur at interband regions, and the genetically complex DNA segments that are inserted generate only a single detectable band.     

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.     

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.     

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.     

Purification of commercial preparations of NADP+ from AMP contamination

A method for purification of commercial preparations of NADP+ from AMP contamination is described. The purification procedure includes one-step anion-exchange chromatography on Dowex-1 (formate) and results in a highly purified salt-free coenzyme with a yield of 70-80%. The chromatography conditions have been selected allowing for complete separation of AMP from NADP+ in a HCOOH concentration gradient. This is followed by NADP+ elution with 1.5 M HCOOH containing HCOOK at a concentration at which the salt remains in solution during subsequent precipitation and washing of NADP+ with acetone. An addition of HCOOK is necessary to reduce the coenzyme elution volume that is important for further precipitation of NADP+ with acetone.     

Short chain dehydrogenase/reductase rdhe2 is a novel retinol dehydrogenase essential for frog embryonic development

The enzymes responsible for the rate-limiting step in retinoic acid biosynthesis, the oxidation of retinol to retinaldehyde, during embryogenesis and in adulthood have not been fully defined. Here, we report that a novel member of the short chain dehydrogenase/reductase superfamily, frog sdr16c5, acts as a highly active retinol dehydrogenase (rdhe2) that promotes retinoic acid biosynthesis when expressed in mammalian cells. In vivo assays of rdhe2 function show that overexpression of rdhe2 in frog embryos leads to posteriorization and induction of defects resembling those caused by retinoic acid toxicity. Conversely, antisense morpholino-mediated knockdown of endogenous rdhe2 results in phenotypes consistent with retinoic acid deficiency, such as defects in anterior neural tube closure, microcephaly with small eye formation, disruption of somitogenesis, and curved body axis with bent tail. Higher doses of morpholino induce embryonic lethality. Analyses of retinoic acid levels using either endogenous retinoic acid-sensitive gene hoxd4 or retinoic acid reporter cell line both show that the levels of retinoic acid are significantly decreased in rdhe2 morphants. Taken together, these results provide strong evidence that Xenopus rdhe2 functions as a retinol dehydrogenase essential for frog embryonic development in vivo. Importantly, the retinol oxidizing activity of frog rdhe2 is conserved in its mouse homologs, suggesting that rdhe2-related enzymes may represent the previously unrecognized physiologically relevant retinol dehydrogenases that contribute to retinoic acid biosynthesis in higher vertebrates.     

Localization and characteristics of DNA underreplication zone in the 75C region of intercalary heterochromatin in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> polytene chromosomes

In Drosophila polytene chromosomes, regions of intercalary heterochromatin are scattered throughout the euchromatic arms. Here, we present data on the first fine analysis of the individual intercalary heterochromatin region, 75C1-2, located in the 3L chromosome. By using electron microscopy, we demonstrated that this region appears as three closely adjacent condensed bands. Mapping of the region on the physical map by means of the chromosomal rearrangements with known breakpoints showed that the length of the region is about 445 kb. Although it seems that the SUUR protein binds to the whole 75C1-2 region, the proximal part of the region is fully polytenized, so the DNA underreplication zone is asymmetric and located in the distal half of the region. Finally, we speculate that intercalary heterochromatin regions of Drosophila polytene chromosomes are organized into three different types with respect to the localization of the underreplication zone.