. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 346 J. Y. BRADFIELD KT AL kb 95- II 24- 03- Figure 1. Total RNA samples (5 ng/\ane) from mid-reproductive cycle Penaeus rannamei tissues were denatured, separated by electro- phoresis in agarose, transferred to nylon membrane, and hybridized with the cloned 3 kb ovarian cDNA. Lane 1, ovary; lane 2, hepatopan- creas; lane 3, muscle. RNA size markers are indicated at left. experiments. (2) Incubation of extracts of various con- centrations of pre-vitellogenic ovaries and nearly mature ovaries with various concentrati
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 346 J. Y. BRADFIELD KT AL kb 95- II 24- 03- Figure 1. Total RNA samples (5 ng/\ane) from mid-reproductive cycle Penaeus rannamei tissues were denatured, separated by electro- phoresis in agarose, transferred to nylon membrane, and hybridized with the cloned 3 kb ovarian cDNA. Lane 1, ovary; lane 2, hepatopan- creas; lane 3, muscle. RNA size markers are indicated at left. experiments. (2) Incubation of extracts of various con- centrations of pre-vitellogenic ovaries and nearly mature ovaries with various concentrations of fusion protein. This experiment demonstrated that the fusion polypep- tide and immunoreactive native ovarian protein were adsorbed to the incubation wells with equal efficiency. We assumed that the common epitopes on the two different molecules (, fusion protein and ovarian poly- peptide) behaved identically in the ELISA. Results Transcript characterization Northern hybridization was performed to determine (1) the size of the highly expressed transcript(s) repre- sented by the cloned ovarian cDNA and (2) which tissues expressed transcripts for production of the major ovarian polypeptide. The cDNA hybridized with a single 6 kb transcript from a mid-cycle ovary (oocyte diameter ~ 150 nm) (Fig. 1). The cDNA did not hybridize with RNA from muscle or hepatopancreas of the same animal. Genetically engineered fusion polypeptide A genetically engineered polypeptide consisting of shrimp ovarian polypeptide and bacterial 0-galactosi- dase was generated for subsequent immunological iden- tification of the shrimp ovarian cDNA translation prod- uct. For generation of this fusion polypeptide, the 3 kb cloned cDNA representing the 6 kb ovarian transcript was inserted into plasmid pUR 291 (Rutherand Miiller- Hill, 1983). This recombinant construct was used for high-level expression of the fusion polypeptide in bacte- ria. The fusion polypeptide consisted of ~ 115 kDa j3- galactosida
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
