DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome.

Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A.     

Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase.

Vaccinia virus encapsidates a DNA-dependent ATPase known as nucleoside triphosphate phosphohydrolase I (NPH I). A bacteriophage lambda gt11 expression library of poxvirus DNA was screened with antibodies specific for NPH I. Positive clones were used to probe restriction fragments of vaccinia virus genomic DNA to locate the NPH I gene. The identity of the open reading frame (ORF) was confirmed by placing it downstream of a bacteriophage T7 promoter, transcribing the ORF in vitro, and translating the RNA in a reticulocyte lysate. A polypeptide of the correct molecular weight, which was recognized by anti-NPH I antibody, was synthesized. Inspection of the deduced amino acid sequence of the NPH I ORF revealed consensus ATP-binding sites.     

Hynes pharyngoplasty revisited

The treatment of velopharyngeal incompetence remains unsatisfactory because the causes are many, as are the variations in anatomic and physiologic defects. Therefore, full assessment and investigation are essential in tailoring the surgery to the defect. A modified Hynes pharyngoplasty has been used in 40 patients, aged 4 to 52, over a 4-year period for velopharyngeal incompetence of varying etiologic causes. Speech was assessed before and at least 6 months after pharyngoplasty. At the same time, radiologic and, when possible, nasendoscopic investigations were undertaken. Thirty-eight patients had no or variable nasal escape (variable defined as achieving intermittent closure), whereas 33 had normal or slight hyponasal resonance. There was only one complication, an asymptomatic dehiscence of the "bucket handle" flap from the posterior wall. Thirteen patients had an assortment of side effects, none requiring surgical treatment. We believe that patients who are suitable for the described sphincter pharyngoplasty are those with slight or moderate nasal escape having a mobile palate with an anteroposterior gap of 5 mm or less.     

Studies on orthocephalization. 10. Behaviour of the visceral part of the rat head during the first 14 days after gestation

The present paper considers the significance of interosseous flexions of the palatal complex in the process of orthocephalization of the rat skull between birth and 7 d p.n. The study is based on a sample of 90 rats divided into 4 age groups, i.e. 0, 4, 7, and 14 d. These rats have been X-rayed, and their photographs subsequently analysed. During the studied period, the constituents of the bony palate, i.e. the horizontal part of the palatine bone, the palatal process of maxilla and the palatal part of premaxilla, increase markedly in length, but with individual differences in growth rate. There is, in the period, a marked decrease in angulation between the cranial base and the palatal plane. This means that the rat skull becomes more orthocranial. There is also a straightening (orthopalatalization) of the palate, as the angle between maxilla and premaxilla becomes more obtuse, and a marked decrease in angulation between the palatine bone and the cranial base. The patterns of angular changes suggest that the process of orthocephalization in the period between birth and 14 d p.n. primarily is a result of an upwards rotation of the palatine bone relative to the cranial base, while interosseous deflections in the palate only play a minor role.     

Standardized method for evaluation of hand disinfection by surgical scrub formulations

A standardized protocol for the evaluation of hand disinfection by surgical scrub formulations was applied to volunteers in a multicenter trial. Povidone iodine (PVI), chlorhexidine (CHX), and a nonmedicated soap (NMS) were tested. The scrubbing procedure involved three daily hand washings for five consecutive days; surviving bacteria were counted daily after being collected in a suitable neutralizing solution. Immediate efficacy (IE), cumulative efficacy (CE), and remanent effect (RE) were calculated by reference to the control hand. Statistical analyses of IE, CE, and RE showed significant differences among the three scrub formulations. IEs of PVI and CHX were equivalent and different from IE of NMS; CE and RE of CHX were higher than those of PVI and NMS. On the basis of the statistical analysis, the population size required for further studies aimed at detecting significant differences between surgical scrub formulations could be estimated.     

Discrimination of jittered sonar echoes by the echolocating bat,Eptesicus fuscus: The shape of target images in echolocation

1. Behavioral experiments with jittering echoes examined acoustic images of sonar targets in the echolocating bat, Eptesicus fuscus, along the echo delay or target range axis. Echo phase, amplitude, bandwidth, and signal-to-noise ratio were manipulated to assess the underlying auditory processes for image formation. 2. Fine delay acuity is about 10 ns. Calibration and control procedures indicate that this represents temporal acuity rather than spectral discrimination. Jitter discrimination curves change in phase when the phase of one jittering echo is shifted by 180 degrees relative to the other, showing that echo phase is involved in delay estimation. At an echo detectability index of about 36 dB, fine acuity is 40 ns, which is approximately as predicted for the delay accuracy of an ideal receiver. 3. Compound performance curves for 0 degrees and 180 degrees phase conditions match the crosscorrelation function of the echoes. The locations of both 0 degrees and 180 degrees phase peaks in the performance curves shift along the time axis by an amount that matches neural amplitude-latency trading in Eptesicus, confirming a temporal basis for jitter discrimination.     

Systematic binding analysis of the insulin gene transcription control region: insulin and immunoglobulin enhancers utilize similar transactivators.

The 5' regulatory region (-345 to +1) of the rat insulin I gene (Ins-I) was examined for binding to cellular factors with short oligodeoxynucleotide probes. Over 40 binding species were detected. The binding profiles were specific for each cell type studied. We characterized the factors binding two elements crucial for enhancer activity (the Nir and Far boxes) which bear sequence similarity to the microE1, microE2, and microE3 elements of the immunoglobulin heavy-chain enhancer. The Nir box binds three cellular factors that display preferential affinities for microE1, microE2, or microE3, and the Far box binds two factors related to microE2 or microE3. The insulin gene enhancer was mutated at the Nir box element to reflect the sequences of microE1, microE2, or microE3. Ins-microE2 was fully active, Ins-microE3 was partially active, and Ins-microE1 was inactive. Thus, factors similar or identical to nuclear factor NF-microE1, NF-microE2, or NF-microE3 may play a role in the activity of the insulin gene enhancer.     

Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein.

Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.