Microtubules are involved in localizing the bicoid RNA to the anterior pole of the oocyte.<20). Here we consider the developmental and/or cellular significance of the localization of these RNAs per se. In Drosophila, both genetic and molecular analyzes have revealed the biological significance of the localization of a number of RNAs in the oocyte and early embryo. <35).
Multiple steps in the localization of bicoid RNA to the anterior pole of the Drosophila oocyte. In some of these embryos there was also a dorsal-ventral difference in the concentration of the RNA (Fig. 7B-D). COl RNA: Mitochondria are uniformly distributed in the cytoplasm of the early embryo (Akiyama and Okada, 1992; Ding et al. 1993c.
We therefore suspect that the COl RNA is not localized in the cytoplasm of the early embryo, but rather is present within the. 16S Large rRNA: The localization of the 16S rRNA to the posterior pole of the early embryo has been studied in detail (Ding et al. 1993c). Localization of 16S rRNA is dependent on components of the posterior polar plasma (Ding et al. 1993c).
By the syncytial blastoderm stage, Hsp83 RNA can only be detected in the polar cells (arrowhead) and a small area of the somatic.
APPENDIX
The conversion strategy used here is based on the site-specific recombination system of the phage PI (Sternberg et al.). First, restriction sites to be placed in the multiple cloning site of the completed vector must be removed. The plasmid portions of the A.EXLX vectors consist of: (1) the 17 elements of pET3xa (the T7 promoter, the coding sequences for the amino terminal end of the product of the T7 gene]0, and the 17 transcription terminator) (Rosenberg et al. 1987); .
The construction of these plasmids, pMP3(+) and pMP3(-), which differ only in the orientation of the f1 ori, is shown in the diagram in Fig. Each of these was tested in each of the constructions to ensure that they are intact and functional. Simon (pers. comm.), where the eDNA can be directionally cloned with all the internal sites protected (see section [g] below for details).
One of the clones was found to be identical to the nematode hsp70A gene (Snutch et al. 1988) over the 187 nt sequence determined. In the case of AEXLX and ASHLX, one would ligate a linker containing an EcoRI site flanked by the last 4 nt of the HindIII recognition site. The eDNA will then be digested with EcoRI+Hindiii and cloned into the unique EcoRI and Hindiiii sites in any of the four vectors we have constructed.
The use of the A.EXLX vectors for gene]0 fusion protein expression requires that the gene/0 and eDNA ORFs are in the same frame. In the case of the A.EXLX vectors, ss DNA (purified as detailed in sections e[ii] and [iii]. Two methods have been developed for using eDNA libraries to generate the large quantities of the ss DNA required for subtractive hybridization procedures (Palazzolo and Meyerowitz 1987; Pruitt 1988).
A detailed map of A.LOX is shown, with the names and locations (±10 bp) of the above restriction sites. Plasmids pMP2(+) and pMP2(−) were introduced into mA.J phage (provided by M. Strathmann and M. Simon) by homologous recombination with a fragment of E. The directions of transcription and gold orientation are represented by arrows.
EXL X ±
APPENDIX
Nonyl acridine orange staining reveals that mitochondria are uniformly distributed in the cortical cytoplasm of the early embryo; thus, the high concentration of 16S RNA at the posterior pole is not due to the high concentration of mitochondria in the posterior polar plasma. Here we report that, consistent with its possible role in whole cell formation, 16S RNA is highly concentrated at the posterior pole of the embryo immediately after fertilization. In addition, this posterior localization of 16S RNA depends on genes required for polar plasma integrity and full cell formation, but.
A much higher level of 16S RNA accumulates at the posterior end of the oocyte some time between stage 12 of oogenesis and egg deposition shortly after fertilization (Figure 2A). However, the timing of posterior localization of 16S RNA is similar to that of the germline transcript, which becomes posterior. By the time pole cell buds appear, the intensity of 16S RNA at the posterior end of the embryo has dropped significantly, becoming only slightly higher than elsewhere in the embryo (Figure 2B).
A second possible explanation for the high posterior concentration of 16S RNA is that the activity of mitochondria in the posterior polar plasma is significantly higher than in the rest of the embryo. Thus, components of the polar plasma, possibly the polar granules per se, are required to establish and/or maintain the high posterior concentration of the 16S RNA in early embryos. Posterior localization of the 16S RNA does not correlate with mitochondrial distribution or activity in early embryos.
This raises the possibility that posterior concentration of the 16S RNA reflects one facet of the association between polar granules and mitochondria (see below). These data suggested that the 16S RNA is an extra-mitochondrial component of the polar plasma that functions in polar cell formation (Kobayashi and Okada 1989). Our RNA tissue in situ analyzes of the 16S and ND-1 RNAs support the hypothesis that 16S RNA is an extra-mitochondrial component of the posterior polar plasma.
The 16S RNA is concentrated in the polar plasma and forms a dense cap at the posterior pole of early embryos, a pattern similar to that of RNAs likely to be components of the polar plasma (such as transcripts of nanos , oskar , and germ cells -less) (Wang and Lehmann 1991; Ephrussi et al. 16S RNA is one of the most abundant polyadenylated RNA species in the early embryo. When pole cells are formed, there is only slightly more RNA at the posterior end than the rest of the de Foster.
The pole cell buds (arrowhead) are not stained. C) In syncytial blastoderm, 16S RNA is uniformly distributed throughout the somatic part of the embryo. Posterior localization of 16S RNA is normal in early cleavage stage embryos produced by females mutant for nanos (A) or pumilio (B), which eliminate components of the posterior polar plasma without affecting the integrity of the polar granules.
