Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.     

Characterization of surface alloantigens of murine neutrophils

Serological analysis of highly purified (>97%) mouse peritoneal exudate neutrophils using a protein-A rosetting technique, showed that these cells possessed the surface phenotype: Ig^–, Thy-1^–, Ly-1^–, Ly-2^–, Ly-3^–, Ly-4⁺, Ly-5⁺, Ly-6⁺, Ly-7^–, Ia^–, FcR⁺ and C3R⁺.     

New mechanical aid to physiotherapy in cystic fibrosis.

The force of impact and frequency of percussion by physiotherapists and parents of children with cystic fibrosis were analysed on a special test rig and incorporated in a prototype percussor. In adult volunteers and cadavers a maximum intrathoracic pressure could be achieved by a critical frequency of mechanical percussion which was higher than that reached by physiotherapists and parents. Consequently the percussor was redesigned to operate at this optimum frequency. It was then discovered that if the percussor was pressed firmly enough against the chest, this maximum intrathoracic pressure could be indicated by quivering of the voice. In a continuing study of intrathoracic pressures obtained mechanically and manually the Salford percussor''s produced higher pressures than the physiotherapists'' and maintained them constantly, while the physiotherapists'' efforts and results varied from one to another. Hospital and domiciliary use of the percussor have shown it to help in the first stage of the physiotherapy routine for patients with cystic fibrosis. The percussor should enable adolescents and adults to treat themselves and encourage twice-daily and more effective chest treatments. It is easy to apply and its speed and efficiency should enable parents to improve the quality of their chest therapy for younger children at home. Its long-term benefits are difficult to assess because of the nature of the disease.     

Chemoimmunotherapy of advanced breast cancer: prolongation of remission and survival with BCG.

Forty-five patients with disseminated breast cancer were given a trial of combination chemotherapy consisting of fluorouracil, adriamycin, and cyclophosphamide (FAC) and immunotherapy with BCG given by scarification. The results were compared with those in a comparable group of 44 patients treated with FAC alone immediately before the chemoimmunotherapy study. The remission rates (73% and 76% for FAC and FAC-BCG respectively) were similar in both studies. The durations of remission for patients on FAC-BCG (medium 12 months) were longer than remissions achieved for patients given FAC alone (median 8 months) (P = 0.068). The most notable effect of BCG was on survival. Thus 21 out of 34 patients achieving remission on FAC-BCG were alive at the time of the last follow-up examination (median over 22 months) compared with 11 out of 32 patients achieving remission on FAC (median 15 months) (P = 0.01). Twenty-six of the 45 patients given FAC-BCG were alive at the time of the last follow-up examination (median over 22 months) compared with 12 of the 44 patients given FAC (median 15 months) (P = 0.005). Although the apparent benefit of BCG could be explained by a maldistribution of some prognostic factors, the data suggest that further trial of chemoimmunotherapy of breast cancer should be carried out.     

Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

The essential mammalian gene TACC3 is frequently mutated and amplified in cancers and its fusion products exhibit oncogenic activity in glioblastomas. TACC3 functions in mitotic spindle assembly and chromosome segregation. In particular, phosphorylation on S558 by the mitotic kinase, Aurora-A, promotes spindle recruitment of TACC3 and triggers the formation of a complex with ch-TOG-clathrin that crosslinks and stabilises kinetochore microtubules. Here we map the Aurora-A-binding interface in TACC3 and show that TACC3 potently activates Aurora-A through a domain centered on F525. Vertebrate cells carrying homozygous F525A mutation in the endogenous TACC3 loci exhibit defects in TACC3 function, namely perturbed localization, reduced phosphorylation and weakened interaction with clathrin. The most striking feature of the F525A cells however is a marked shortening of mitosis, at least in part due to rapid spindle assembly. F525A cells do not exhibit chromosome missegregation, indicating that they undergo fast yet apparently faithful mitosis. By contrast, mutating the phosphorylation site S558 to alanine in TACC3 causes aneuploidy without a significant change in mitotic duration. Our work has therefore defined a regulatory role for the Aurora-A-TACC3 interaction beyond the act of phosphorylation at S558. We propose that the regulatory relationship between Aurora-A and TACC3 enables the transition from the microtubule-polymerase activity of TACC3-ch-TOG to the microtubule-crosslinking activity of TACC3-ch-TOG-clathrin complexes as mitosis progresses. Aurora-A-dependent control of TACC3 could determine the balance between these activities, thereby influencing not only spindle length and stability but also the speed of spindle formation with vital consequences for chromosome alignment and segregation.