DNA primase isolated from human mitochondria sediments in glycerol density gradients at 30S and 70S. These unusually high sedimentation coefficients are a result of association of the primase activity with RNA. Treatment of primase with nuclease not only affects its sedimentation behavior, but also inactivates the primase activity. The major RNA species that cofractionates with primase activity is shown by direct sequence analysis to be cytosolic 5.8S ribosomal RNA (rRNA). Specific degradation of endogenous 5.8S rRNA using ribonuclease H and oligonucleotides complementary to 5.8S rRNA results in reduction of primase activity. Other small RNAs may play a structural role in the formation of an active DNA primase complex.

We show that the germline specificity of P element transposition is controlled at the level of mRNA splicing and not at the level of transcription. In the major P element RNA transcript, isolated from somatic cells, the first three open reading frames are joined by the removal of two introns. Using in vitro mutagenesis and genetic analysis we demonstrate the existence of a third intron whose removal is required for transposase production. We propose that this intron is only removed in the germline and that its removal is the sole basis for the germline restriction of P element transposition.

By discrete manipulation of the endocrine cues that control insect metamorphosis, it has been possible to examine the mechanisms governing the growth of neural processes during development. During the transition from larva to pupa in the hawkmoth, Manduca sexta, identified sensory neurons reorganize their central projections to evoke a new behavior–the gintrap reflex. Topical application of a juvenile hormone analog to the peripheral cell bodies of these sensory neurons during a critical period of development caused them to retain their larval commitment rather than undergo pupal development with the rest of the animal. The sensory neurons retained the larval arborization pattern within the pupal CNS and were unable to evoke the gin-trap reflex. Thus, the hormonal environment of the cell body is critical for controlling growth and synapse formation by distant axonal processes.

Synthesis of human light-strand mitochondrial DNA was accomplished in vitro using DNA primase, DNA polymerase, and other accessory proteins isolated from human mitochondria. Replication begins with the synthesis of primer RNA on a T-rich sequence in the origin stem-loop structure of the template DNA and absolutely requires ATP. A transition from RNA synthesis to DNA synthesis occurs near the base of the stem-loop structure and a potential recognition site for signaling that transition has been identified. The start sites of the in vitro products were mapped at the nucleotide level and were found to be in excellent agreement with those of in vivo nascent light-strand DNA. Isolated human mitochondrial enzymes recognize and utilize the bovine, but not the mouse, origin of light-strand replication.

Faithful transcription of human mitochondrial DNA has been reproduced in vitro, using a fraction of mitochondrial proteins capable of accurate initiation at both the heavy- and light-strand promoters. Here we report the initial dissection of this system into two nonfunctional components which, upon mixing, reconstitute promoter-specific transcriptional capacity in vitro. One of these components copurifies with the major nonspecific RNA polymerase activity, suggesting its identity. The other component lacks significant polymerase activity, but contains a protein or proteins required for accurate initiation at the two individual promoters by isolated mitochondrial RNA polymerase. This factor facilitates specific transcription, but has little or no effect on nonspecific transcription of a synthetic copolymer (poly(dA-dT)), indicating a positive role in proper promoter recognition. The transcription factor markedly stimulates light-strand transcription, but only moderately enhances transcription initiation at the heavy-strand promoter, suggesting different or additional factor requirements for heavy-strand transcription. These requirements may reflect the functional differences between heavy- and light-strand transcription in vivo and, in particular, the role of the light-strand promoter in priming of heavy-strand DNA replication.

Mammalian mitochondrial DNA maintains a novel displacement-loop region containing the major sites of transcriptional initiation and the origin of heavy strand DNA replication. Because the exact map positions of the 5’ termini of nascent mouse displacement-loop strands are known, it is possible to examine directly a potential relationship between replication priming and transcription. Analyses of in vivo nucleic acids complementary to the displacement-loop region reveal two species with identical 5’ ends at map position 16 183. One is entirely RNA and the other is RNA covalently linked to DNA. In the latter the transition from RNA to DNA is sharp, occurring near or within a series of previously identified conserved sequences 74-163 nucleotides downstream from the transcriptional initiation site. These data suggest that the initial events in replication priming and transcription are the same and that the decision to synthesize DNA or RNA is a downstream event under the control of short, conserved displacement-loop template sequences.

Using a novel method for detecting cross-homologous nucleic acid sequences we have isolated the gene coding for the major rhodopsin of Drosophila melanogaster and mapped it to chromosomal region 92B8-11. Comparison of cDNA and genomic DNA sequences indicates that the gene is divided into five exons. The amino acid sequence deduced from the nucleotide sequence is 373 residues long, and the polypeptide chain contains seven hydrophobic segments that appear to correspond to the seven transmembrane segments characteristic of other rhodopsins. Three regions of Drosophila rhodopsin are highly conserved with the corresponding domains of bovine rhodopsin, suggesting an important role for these polypeptide regions.

Many of the genes in the regulatory hierarchy controlling sex determination in Drosophila melanogaster are known. Here we examine how this regulatory hierarchy controls the expression of the structural genes encoding the female-specific yolk polypeptides. Temperature shift experiments with a temperature-sensitive allele of the sex determination regulatory gene transformer-2 (tra-2) showed that tra-2+ function is required in the adult for both the sex-specific initiation and maintenance of YP synthesis. Control of the YP genes by this regulatory hierarchy is at the level of transcription, or transcript stability. The results of temperature shift experiments with abdomens isolated from tra-2ts homozygotes support the notion that the tra-2+ function acts in a cell-autonomous manner to control YP synthesis. These results provide a paradigm for the way this regulatory hierarchy controls the terminal differentiation functions for sexually dimorphic development.

We have made a P-element derivative called Pc[ry], which carries the selectable marker gene rosy, but which acts like a nondefective, intact P element. It transposes autonomously into the germline chromosomes of an M-strain Drosophila embryo and it mobilizes in trans the defective P elements of the singed-weak allele. Frameshift mutations introduced into any of the four major open reading frames of the P sequence were each sufficient to eliminate the transposase activity, but none affected signals required in cis for transposition of the element. Complementation tests between pairs of mutant elements suggest that a single polypeptide comprises the transposase. We have examined transcripts of P elements both from natural P strains and from lines containing only nondefective Pc[ry] elements, and have identified two RNA species that appear to be specific for autonomous elements.

The major site of in vivo transcriptional initiation for both heavy and light strands of human mitochondrial DNA is the displacement-loop region. Transcripts synthesized in vitro by human mitochondrial RNA polymerase were mapped to the nucleotide level and have identical 5’ end map positions to those reported for in vivo primary transcripts. An ordered series of deletion clones, whose template sequences were truncated at either the 5’ or 3’ end, was used to identify the precise mitochondrial DNA sequence required for initiation of transcription. The data provide a definitive assignment of the promoter for heavy-strand transcription occurring within -16 to +7 of the transcriptional start site 16 nucleotides upstream of the 5’ end of the gene for tRNAPhe and of the promoter for light-strand transcription occurring within -28 to +16 of the transcriptional start site at the 5’ end of "7S RNA." Within each control sequence is a candidate promoter whose consensus sequence is 5’-CANACC(G)CC(A)AAAGAPyA-3’ and in both cases transcriptional initiation occurs within six to eight nucleotides of the 3’ end of this sequence. The transcriptional start site is an integral part of each promoter and each promoter can function in the absence of the other.