Infection of the germ line by retroviral particles produced in the follicle cells: a possible mechanism for the mobilization of the gypsy retroelement of Drosophila.

The gypsy retroelement of Drosophila moves at high frequency in the germ line of the progeny of females carrying a mutation in the flamenco (flam) gene. This high rate of de novo insertion correlates with elevated accumulation of full-length gypsy RNA in the ovaries of these females, as well as the presence of an env-specific RNA. We have prepared monoclonal antibodies against the gypsy Pol and Env products and found that these proteins are expressed in the ovaries of flam females and processed in the manner characteristic of vertebrate retroviruses. The Pol proteins are expressed in both follicle and nurse cells, but they do not accumulate at detectable levels in the oocyte. The Env proteins are expressed exclusively in the follicle cells starting at stage 9 of oogenesis, where they accumulate in the secretory apparatus of the endoplasmic reticulum. They then migrate to the inner side of the cytoplasmic membrane where they assemble into viral particles. These particles can be observed in the perivitelline space starting at stage 10 by immunoelectron microscopy using anti-Env antibodies. We propose a model to explain flamenco-mediated induction of gypsy mobilization that involves the synthesis of gypsy viral particles in the follicle cells, from where they leave and infect the oocyte, thus explaining gypsy insertion into the germ line of the subsequent generation.

An env-like protein encoded by a Drosophila retroelement: evidence that gypsy is an infectious retrovirus.

The gypsy element of Drosophila differs from most LTR retrotransposons in containing a third open reading frame that resembles retroviral env genes. The protein encoded by ORF3 is glycosylated and processed, like all retroviral envelope proteins. The protein is expressed at high levels in fly strains in which gypsy elements are active. In these strains the protein is found primarily in viral particles. When larvae of fly strains in which gypsy is normally inactive are exposed to sucrose gradient fractions containing these particles, a high level of gypsy insertion activity is observed in their progeny. Thus, gypsy has the expected properties of an insect retrovirus.

P element-mediated in vivo deletion analysis of white-apricot: deletions between direct repeats are strongly favored.

We have isolated and characterized deletions arising within a P transposon, P[hswa], in the presence of P transposase. P[hswa] carries white-apricot (wa) sequences, including a complete copia element, under the control of an hsp70 promoter, and resembles the original wa allele in eye color phenotype. In the presence of P transposase, P[hswa] shows a high overall rate (approximately 3%) of germline mutations that result in increased eye pigmentation. Of 234 derivatives of P[hswa] with greatly increased eye pigmentation, at least 205 carried deletions within copia. Of these, 201 were precise deletions between the directly repeated 276-nucleotide copia long terminal repeats (LTRs), and four were unique deletions. High rates of transposase-induced precise deletion were observed within another P transposon carrying unrelated 599 nucleotide repeats (yeast 2 mu FLP; recombinase target sites) separated by 5.7 kb. Our observation that P element-mediated deletion formation occurs preferentially between direct repeats suggests general methods for controlling deletion formation.

Polyadenylylation in copia requires unusually distant upstream sequences.

Retroviruses and related genetic elements generate terminally redundant RNA products by differential polyadenylylation within a long terminal repeat. Expression of the white-apricot (wa) allele of Drosophila melanogaster, which carries an insertion of the 5.1-kilobase retrovirus-like transposable element copia in a small intron, is influenced by signals within copia. By using this indicator, we have isolated a 518-base-pair deletion, 312 base pairs upstream of the copia polyadenylylation site, that is phenotypically like much larger deletions and eliminates RNA species polyadenylylated in copia. This requirement of distant upstream sequences for copia polyadenylylation has implications for the expression of many genetic elements bearing long terminal repeats.

Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli.

Respiratory deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G59 are pleiotropically deficient in respiratory chain components and in mitochondrial ATPase. This phenotype has been shown to be a consequence of mutations in a nuclear gene coding for mitochondrial leucyl-tRNA synthetase. The structural gene (MSL1) coding for the mitochondrial enzyme has been cloned by transformation of two different G59 mutants with genomic libraries of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein of 894 residues with a molecular weight of 101,936. The amino-terminal sequence (30-40 residues) has a large percentage of basic and hydroxylated residues suggestive of a mitochondrial import signal. The cloned MSL1 gene was used to construct a strain in which 1 kb of the coding sequence was deleted and substituted with the yeast LEU2 gene. Mitochondrial extracts obtained from the mutant carrying the disrupted MSL1::LEU2 allele did not catalyze acylation of mitochondrial leucyl-tRNA even though other tRNAs were normally charged. These results confirmed the correct identification of MSL1 as the structural gene for mitochondrial leucyl-tRNA synthetase. Mutations in MSL1 affect the ability of yeast to grow on nonfermentable substrates but are not lethal indicating that the cytoplasmic leucyl-tRNA synthetase is encoded by a different gene. The primary sequence of yeast mitochondrial leucyl-tRNA synthetase has been compared to other bacterial and eukaryotic synthetases. Significant homology has been found between the yeast enzyme and the methionyl- and isoleucyl-tRNA synthetases of Escherichia coli. The most striking primary sequence homology occurs in the amino-terminal regions of the three proteins encompassing some 150 residues. Several smaller domains in the more internal regions of the polypeptide chains, however, also exhibit homology. These observations have been interpreted to indicate that the three synthetases may represent a related subset of enzymes originating from a common ancestral gene.

Reliability of antenatal testing: estriol levels versus nonstress testing.

During the third trimester of pregnancy, 334 high-risk patients were followed with antenatal fetal heart rate (FHR) evaluation and with serial determinations of unconjugated plasma estriol. The antenatal FHR tests included nonstress testing (NST) and investigation of beat-to-beat variability. Data indicate that the NST was more reliable than estriol analysis in assessing fetal compromise. While predictive values of negative test results did not differ statistically, the NST/beat-to-beat assessment was particularly accurate in identifying fetal jeopardy in more than 45.0% of the fetuses at risk, whereas only 22.8% cases of jeopardy were accurately predicted by abnormal estriol values. An abnormal NST with loss of beat-to-beat FHR variability should therefore take precedence over plasma estriol determinations during antenatal surveillance of high-risk obstetric patients.

Implications for multiple transmitter mediation of amphetamine-induced stereotypies.

Gerbils were pretreated with the dopamine (DA) receptor blocker, pimozide, prior to stereotypy-inducing injections of d-amphetamine. Some of the stereotypies induced with amphetamine were blocked, but some were not, supporting the hypothesis that multiple neurotransmitters are involved in the mediation of amphetamine-induced stereotypy. In addition, when apomorphine hydrochloride was injected, different stereotypic motor behaviors were induced than were induced with amphetamine. The behavioral changes following amphetamine treatment could be classified into four groups: (1) those that are probably DA related, based on the fact that they were induced with either amphetamine or apomorphine, and amphetamine induction was blocked with pimozide; (2) those that are probably not DA related because they were not induced by apomorphine, and amphetamine induction was not blocked by pimozide; (3) those behaviors that may be incompatible with stereotypic behaviors, were reduced with either amphetamine or apomorphine, but were not maintained with pimozide; and (4) circling, which was induced with amphetamine, blocked with pimozide, but not induced with apomorphine.

A new iud insertion technique utilizing cervical priming with prostaglandin.

Cervical priming with the aid of a single 15-ME-PGF2a vaginal suppository prior to IUD insertion resulted in cervical changes which facilitated the procedure. A 0.5 mg 15(S)-15-prostaglandin F2a methyl ester vaginal suppository was administered one hour prior to the IUD insertion in all patients studied. The insertion was performed in all patients studied. The insertion was performed from seven to seventeen days following the LMP with the exception of four patients with prolonged amenorrhea. A mean increase in cervical dilatation of 2.14 mm was achieved with minimal side effects. The cervical ripening and dilatation produced by the suppository increased the ease of IUD insertion , and expanded the time frame in which an IUD insertion could be performed. The method was well tolerated by all patients and eliminated the nausea and syncope often associated with IUD insertion.

A new modality in nonstress testing: evaluation of beat-to-beat fetal heart rate variability.

Fetal monitoring equipment that provided accurate external measurement of the interval between each fetal heartbeat permitted the evaluation of beat-to-beat fetal heart rate (FHR) variability as part of routine nonstress testing. Nonstress tests (NSTs) were performed on 350 high-risk patients over a 12-month period. The beat-to-beat FHR variability and the reactivity of the last NST within 7 days of labor were analyzed in relation to the appearance of fetal distress during labor as indicated by late decelerations. Beat-to-beat FHR variability combined with nonstress testing was more predictive of subsequent fetal distress than nonstress testing alone. In all instances, complete loss of beat-to-beat FHR was followed by fetal distress during labor. Fetal distress was present in only 39% of labors following nonreactive NSTs. The inclusion of beat-to-beat FHR variability in nonstress testing can significantly aid in the detection of fetal compromise.