Clones containing DNA uniquely transcribed during oogenesis were isolated from genomic DNA phage libraries for the two related Dipteran flies C. erythrocephala and D. melanogaster. Poly(A)$\sp{+}$ RNA from early or mid-stage ovarian follicles of C. erythrocephala was used to generate radiolabelled oogenesis-specific cDNA probes for screening the phage libraries. A cDNA probe made from mid-stage embryo poly(A)$\sp{+}$ RNA was used as the differential screening probe. Thus plaques hybridizing to the two oogenesis-specific probes but not the mid-stage embryo probe were selected as potentially containing oogenesis-specific genes. Two further rounds of screening were used to eliminate false positives and, after plaque purification, restriction digests of the remaining clones were screened by Southern blot hybridization to identify DNA fragments transcribed in an oogenesis-specific manner. To date, 22 oogenesis-specific clones have been isolated from C. erythrocephala and two from D. melanogaster by this method.
In situ hybridization to sections of ovarian follicles has been used to determine the cell types within the follicles in which the various genes are expressed. Radiolabelled RNA probes for four of the C. erythrocephala oogenesis-specific clones and the two D. melanogaster clones have been hybridized to ovarian follicles. Two of the C. erythrocephala clones and both of the D. melanogaster clones have thus been shown to be transcribed in the germ-like cells of the follicles as opposed to the somatic follicle cells.
Further studies have been concentrated on the two germ-line transcribed, oogenesis-specific clones isolated form the D. melanogaster clone library. In situ hybridization of the two D. melanogaster clones to the salivary gland polytene chromosomes has established that one clone is derived from chromosome region 31B/D (clone DA) and the second clone from region 98E/F (clone DM). Region 31B/D is rich in female sterile mutations and detailed genetic mapping of the DA clone and of these mutations was performed (using deficiency chromosomes) to determine which mutations might represent the DA gene. cDNA clones have been isolated for the transcribed region of clone DA and have been used to further define the transcription unit from this region of the D. melanogaster genome.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/16194 |
| Date | January 1988 |
| Creators | Tucker, Mark Allen |
| Contributors | Beckingham, Kathy |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | 111 p., application/pdf |
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