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.     

Regulation of the elemental composition of the epididymal fluids in the tammar, Macropus eugenii

Micropuncture, microanalytical and microelectrode techniques were used to study electrochemical aspects of 7 elements and fluid in the ductuli efferents and ductus epididymidis of the tammar. Rete testis fluid was isosmotic with blood and had a lower pH. It also contained lower concentrations of bicarbonate, sodium, calcium, magnesium, phosphorus and sulphur and higher concentrations of potassium and chloride than blood. The luminal fluid was acidified further during passage through the sperm ducts and all of the elements which were studied moved in or out of the lumen, usually against an electrochemical gradient. The ductuli efferents reabsorbed 87% of the fluid leaving the testis without changing the intraluminal concentrations of sodium, potassium and calcium, but the concentrations of magnesium, phosphorus and sulphur increased. The caput epididymidis reabsorbed about half the fluid entering it: sodium concentrations decreased and those of potassium and phosphorus increased. There was also some fluid reabsorption and an increase in the values of potassium and phosphorus in the corpus epididymidis. There was little net transport of fluid in the cauda epididymidis; sodium, chloride, magnesium and phosphorus concentrations decreased and potassium values increased. Studies involving filtration through a dialysis membrane of blood and fluid from the rete testis and cauda epididymidis showed that, whilst some of the calcium, magnesium, phosphorus and sulphur was associated with high molecular weight compounds in blood, the association was not significant in the reproductive fluids.     

Isolation and cross-sensitivity of X-ray-sensitive mutants of V79-4 hamster cells

The V79-4 Chinese hamster line was mutagenized and surviving clones screened for X-ray sensitivity using a replica microwell technique. One slightly sensitive clone and 3 clearly sensitive clones were isolated from approximately 5000 screened, and designated irs 1 to irs 4. The 3 more sensitive clones showed different responses to the genotoxic agents mitomycin C (MMC), ethyl methanesulphonate (EMS) and ultraviolet light (UV). irs 1 showed considerable sensitivity to all the agents tested, in the order MMC much greater than EMS greater than UV. irs 2 and irs 3 had similar sensitivities to EMS and to UV (EMS greater than UV) but irs 3 was more sensitive than irs 2 to MMC. None of these mutants is identical in phenotype to previously published mutants.     

Optimizing the expression of chimeric genes in plant cells

Summary The Serratia marcescens chiA gene encodes a secreted chitinase activity which contributes to the fungal growth inhibition exhibited by this bacterium. The coding region from the chiA gene was fused to the promoter and 3 polyadenylation region of the Agrobacterium nopaline synthase gene. Site-directed mutagenesis of specific nucleotides surrounding the initiating AUG of the coding sequence of this chimeric gene resulted in up to an eight-fold increase in the amount of chitinase protein detected in transformed plant tissue. Analysis of the chiA mRNA indicated that these nucleotides also affected mRNA levels. At least 50% of the chitinase protein produced in transformed tobacco cells was the same molecular weight as the S. marcescen secreted protein.     

Phosphorus nutrition of jarrah (Eucalyptus marginata) seedlings

Summary Effects of calcium phosphate supply on plant dry matter and phosphorus concentrations of parts of jarrah (Eucalyptus marginata) seedlings grown in a lateritic topsoil from the jarrah forest were examined in two glasshouse trials. Phosphorus deficiency depressed root and shoot dry weights and severely deficient leaves were smal and purple with prominent red major veins. Phosphorus deficiency severely reduced stem phosphorus levels (0.5% to 0.02%, experiment 1). Phosphorus concentrations were higher in bark than wood and the amount of phosphorus in the bark was sensitive to stem age and phosphate supply. Phosphorus adequate plants had bark phosphorus concentrations in the range 0.2–0.9% compared to <0.1% in deficient plants (experiment 2). Jarrah leaves accumulated dry matter up to 80 days after expansion and some leaves exported phosphorus during this period. Bark analysis may therefore be preferable to leaf analysis for detecting phosphorus deficiency in this species.     

Different subfamilies of alphoid repetitive DNA are present on the human and chimpanzee homologous chromosomes 21 and 22.

The alphoid repeat DNA on chimpanzee chromosome 22 was compared with alphoid repeat DNA on its human homologue, chromosome 21. Hybridization of different alphoid probes under various conditions of stringency show that the alphoid repeats of chimpanzee chromosome 22 are not closely related to those of human chromosome 21. Sequence analysis of cloned dimer and tetramer EcoRI fragments from chimpanzee chromosome 22 confirm the low overall level of homology, but reveal the presence of several nucleotide changes which are exclusive to the chromosome 21 subfamily of human alphoid DNA. Southern blot analysis of alphoid repeat DNA on the chimpanzee X chromosome suggests this subfamily has been strongly conserved during and since the separation of chimpanzee and man although the two subfamilies can be distinguished on the basis of Taq I restriction fragments.     

Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells.

The close association of human papillomavirus type 16 DNA with a majority of cervical carcinomas implies some role for the virus in this type of cancer. To define the transforming properties of HPV-16 DNA in vitro we have now performed transfection experiments on baby rat kidney cells using HPV-16 DNA in conjunction with an activated ras gene. We have demonstrated that a 6.6-kb DNA fragment, containing the early genes of HPV-16 under the control of Moloney murine leukaemia virus long terminal repeats (MoMuLV-LTRs), cooperates with EJ-ras in transforming these cells. Both DNAs are required and neither alone is effective. The cooperating activity appears to reside in a protein or proteins derived from the E6/E7 region of the HPV-16 genome.     

Elimination of the non-specific binding of avidin to tissue sections

Summary A simple procedure is described for eliminating non-specific staining with avidin—peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 µg/ml biotinylatedPisum sativum agglutinin. Avidin—peroxidase conjugates (5 µg/ml), diluted in standard 0.05m tris-buffered saline, pH 7.6, containing 0.139m NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125m Tris-buffered saline (containing 0.347m NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.