Analytical studies of chromosomal transcription detected by an endogenous hybridization technique (EHT) using autoradiography and indirect immunofluorescence in Drosophila hydei.
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Biomedical subjects
Publications and source records attributed to C Alonso.
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The maximum grain density over the "heat-shock" locus 2-48BC of Drosophila hydei polytene chromosomes obtained after in situ hybridization of nuclear RNA extracted from tissue culture cells labelled during incubation at 37 degrees C is five times higher than that obtainable by using polysomal RNA isolated from the same cells. Furthermore, the addition of a large excess of unlabelled polysomal RNA reduced the amount of in situ hybridization of nuclear RNA by only 20% showing that nuclear 2-48BC RNA contains sequences not present in polysomal 2-48BC RNA.--The polysomal 2-48BC RNA is polyadenylated, as are the RNA sequences present in the polysomes complementary to the other two major "heat shock" loci 2-32A and 2-36A. Polyadenylated RNA, with an apparent size of 15S, complementary to locus 2-48BC is also found in the cytoplasm of D. hydei salivary glands.
U.V. microspectrophotometry has been used to calculate quantities of nucleic acids and proteins of complete polytene chromosomal sets and specific regions of these chromosomes. It has been found that in chromosomes the ratio of DNA to proteins is approximately 1:4. This ratio however changes when specific regions are compared. The average ratio of DNA to proteins in a puffed region (2-48B4C5) increases to 1:16 in contrast to 1:6 from the same region but in non puffed state. At the same time the RNA quantity increases by a factor of 2. thermal denaturation profiles of formaldehyde fixed chromosomes show that the Tm of this region in puffed and non puffed state differ by 10 degrees C. Moreover these profiles suggest that a large fraction of histone-bound DNA is destabilized during puffing.
[3H]Poly(U) hybridizes very rapidly to polytene DNA from Drosophila hydei. When hybridization is performed at 30 degrees C in 2 X SSC to a large excess of DNA, 95% of the poly(U) becomes ribonuclease resistant. Also, complementary RNA transcribed in vitro from polytene DNA hybridizes to poly(U). 023--0.25% of the DNA is composed of (dA)-rich sequences and 0.23--0.31% of cRNA hybridizes to [3H]poly(U). The length of the (dA)-rich sequences on the DNA and cRNA is 40 nucleotides. The Tm values of these hybrids formed between DNA or cRNA-poly(U) is 45 degrees C. The poly(A) fragments from cytoplasmic RNA ranged from 80 to 170 nucleotides in lenght, and migrated in polyacrilamide gels as a broad peak. The average sizes of the poly(A) fragments from the poly(A)-containing RNA transcribed by nuclei isolated from salivary glands in vivo or in vitro were 40, 70, 170 and 70 nucleotides, respectively. Hybridization in situ of [3H]-poly(U) to chromosome squashes indicated that the (dA)-rich sequences are randomly distributed over the whole genome.
The DNA content of individual subregions along the 4th chromosome of Drosophila hydei has been measured. 51% of the subregions have a DNA content averaging 0.7-0.8 pg; 31% a mean DNA content of 0.25-0.35 pg and 18% a mean DNA content of 1.7 pg. Moreover the structural chromosomal distribution of moderately repetitive DNA is not random since the specific activity of the chromosomal segments in terms of those sequences is not the same. 9% of the subregions are very poor in repetitive sequences and 18% rich in repetitive DNA while being very poor in DNA.
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Serial endocrine studies were performed during the refeeding of a group of nine young women who presented with amenorrhea associated with injudicious dieting. No significant psychopathology was disclosed by the Lanyon Psychological Screening Inventory in any of the patients, six of whom were single and three married and infertile. The mean weight before refeeding was 81.6% of the normal for height. The mean initial serum follicle-stimulating hormone concentration was within the limits of normal for young women of reproductive age, but the serum luteinizing hormone (LH) concentration and the vaginal maturation score were low. As weight was regained, the serum LH concentration increased (the pooled r for LH versus percent normal weight = 0.85) and the vaginal maturation score rose (the pooled r for vaginal maturation score versus percent normal weight = 0.93). When a mean weight of 96.6% of normal was reached after a mean of 5.3 (range, 2 to 11) months of refeeding, spontaneous menses occurred. The initial cycle was ovulatory in two instances and anovulatory in five, as determined by basal body temperature patterns, plasma progesterone concentrations, and endometrial biopsies. With continued weight gain, menstruation became regular and the three married patients conceived.
The location of the 5S ribosomal RNA cistrons in band 2-23B1,2 of the polytene (salivary gland) chromosomes of Drosophila hydei was indicated by in situ hybridization of tritiated low molecular weight RNA fractionated from total in vivo synthesized larval RNA or from in vitro synthesized salivary gland RNA and competition of the hybridization of this RNA by 5S RNA obtained from calf lens ribosomes. -- At the submicroscopic level, band 2-23B1,2 in salivary gland chromosomes shows a compact organization. The adjacent region, 23B2, is slightly puffed and displays typical RNP particles, some of which may be observed close to band 2-23B1,2.
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