Genetic and molecular analysis of spontaneous respiratory deficient (res-) mutants of Escherichia coli K-12

Respiratory deficient (res-) mutants of E. coli are slow growing microcolonial, anaerobic, catalase and benzidine negative strains whose broad phenotypic alteration may result from pleiotropic mutations in genes of the hemin biosynthetic pathway. They are easily recovered from platings of sensitive cells on concentrations of gentamicin higher than the minimal inhibitory concentration. These mutants show a dramatic change in their biochemical diagnostic profile resulting primarily from deficiencies in the active transport mechanisms of the cell. Using well-marked F- and Hfr strains, 157 mutants were analyzed from 3 different parent strains; all but 2 resulted from mutations in 3 loci of the hemin biosynthetic pathway. Of these a marked skew to hemB- mutations was seen, with more than 80% mapping there. The possibility that this hot spot resulted from transpositional activity was tested by Southern hybridization of EcoRI digests of the chromosomal DNA, using as a probe, a 2.8-kb fragment containing the hemB gene. The WT and other hemB+ control strains contained a 14.6-kb fragment. Of 18 hemB strains tested, 14 showed deletion and insertion mutations which fell into four classes based on the variation in the size of the fragment or on the absence of hybridization. The latter resulted from complete deletion of the hemB gene. An increase in fragment size from 1.5-kb to 3.4-kb was observed in some of the strains.     

Multiple uses of liquepipets in Southern, northern, and western blots

Individually wrapped, sterile disposable transfer pipets can be used in the isolation of ds-DNA and ds-RNA fragments from gels as well as in the screening of multiple samples in Southern, Northern, and Western blots without potential contamination by exogenous nucleases and proteases. The sensitivity and results obtained by this method are comparable to those obtained by conventional methods. All the prehybridization, blocking, hybridization, and detection processes can be performed within the transfer pipet. The isotopically labeled probes used in hybridization can easily be recovered, stored for reuse, or disposed of as waste with no potential contamination of personnel or laboratory equipment. Strip blots are stable in appropriate buffers within the liquepipets which can be shipped easily worldwide for comparative analyses by collaborative investigators. This method is simple, time saving, and inexpensive and is particularly suitable for multiple sample screening. Other potential applications of this procedure are discussed.     

Surface properties of Streptococcus salivarius HB and nonfibrillar mutants: measurement of zeta potential and elemental composition with X-ray photoelectron spectroscopy.

To characterize the functional cell surface, the zeta potentials and elemental surface composition of Streptococcus salivarius HB and a range of mutants with known molecular surface structures were determined. Zeta potentials of fully hydrated cells were measured as a function of pH in dilute potassium phosphate solutions, yielding isoelectric points of the strains. Elemental composition (O, C, N, and P) of the outer 2 to 5 nm of the freeze-dried cell surfaces were measured by X-ray photoelectron spectroscopy. An increasing loss of proteinaceous fibrillar surface antigens of the mutants was found to be accompanied by a progressive decrease in the N/C ratio from 0.104 in the parent strain HB to 0.053 in mutant HBC12. Simultaneously, the value of the isoelectric point shifted from 3.0 to 1.3. In a previous study (A.H. Weerkamp, H.C. van der Mei, and J. W. Slot, Infect. Immun. 55:438-455, 1987) on the cell surfaces of the same strains, it was shown that removal of fibrils led to increased exposure of (lipo)teichoic acid at the surface, which explains the low isoelectric point caused by the low pKa of the phosphate groups.     

Comparison between the adhesion to solid substrata of Streptococcus mitis and that of polystyrene particles.

The adhesion of Streptococcus mitis to solid substrata from phosphate suspensions with various ionic strengths was studied and compared with the adhesion of polystyrene particles. At all ionic strengths, the interfacial free energy of adhesion governed the relative number of bacteria or polystyrene particles adhering at equilibrium, except that in a low-ionic-strength buffer, adhesion occurred less frequently because of increased electrostatic repulsion. Large differences between bacterial and polystyrene particle adhesion were observed, as indicated by the ratio of bacteria to polystyrene particles adhering, which decreased from 30 to 4 with a change from low to high ionic strength.     

Proliferative response of seminal vesicle cells to androgen in mice castrated neonatally and pretreated with estrogen or androgen at adulthood

Seminal vesicle cells of neonatally castrated adult mice show poor response to androgen, compared to those of mice castrated at adulthood; effects of pretreatment with androgen or estrogen at adulthood on androgen-induced proliferation of the seminal vesicle cells were examined in neonatally castrated mice. Male mice castrated at day 0 after birth were pretreated with daily injections of testosterone propionate (TP, 100 micrograms/mouse), 17 beta-estradiol (E2, 5 micrograms/mouse) or vehicle for 20 days starting from day 60; daily TP injections (100 micrograms/mouse) for 30 days were started again from day 110 in all the pretreated mice to examine androgen-induced proliferation by incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles. Both TP and E2 pretreatments significantly increased the seminal vesicle weight found before TP treatment. However, androgen-induced proliferation of the seminal vesicle found in neonatally castrated mice (poor response; long duration with a low peak on day 3) was changed at least in part to that found in mice castrated at adulthood (good response; short duration with a high peak on day 3) only following the TP pretreatment but not at all following the E2 pretreatment. The E2 pretreatment induced poor androgen-induced proliferation with a low peak on day 7.     

Urea-induced transformation of native estrogen receptor and evidence for separate DNA-and ATP-binding sites

We have investigated the involvement of hydrophobic receptor domains during transformation of the native estrogen receptor to a form(s) with high affinity for immobilized DNA and ATP. In the presence of 6 M urea the intact estrogen-receptor complex was completely (greater than 90%, n = 12) transformed into a DNA-binding configuration but only partially (35-45%, n = 8) transformed into an ATP-binding state. Similar experiments performed with unliganded receptor preparations further distinguished the receptor's DNA and ATP binding properties. While the urea-induced increase in receptor affinity for DNA-agarose was estrogen-dependent, the urea-induced increase in affinity for ATP-agarose was steroid-independent. This is the first direct evidence that hydrophobic receptor domains may be involved in the steroid-dependent exposure of the DNA binding site. This event is partially reversible and suggests that electrostatic interactions alone may not be sufficient to accurately describe receptor recognition of specific DNA acceptor sites.     

Monoclonal antibody detection of transformation associated protein (TAP) in ts110 Moloney murine sarcoma virus transformed 6M2 cells that are different from the mos gene product

By the use of a highly specific monoclonal antibody (designated MC), we were able to detect three radiolabeled bands with molecular weights of 60,000, 63,000, and 66,000 daltons in the ts-110 Moloney murine sarcoma virus mutant-transformed rat kidney cells known as 6M2. Expression of transformation properties as well as these three bands in 6M2 cells was found to be temperature sensitive. Therefore, MC detected factors that are apparently associated with the transformation of 6M2 cells. These factors are tentatively referred to as transformation associated proteins. These transformation proteins were found in two other Moloney murine sarcoma virus-transformed rat cell lines. These proteins were found to differ from known gene products of the ts-110 Moloney murine sarcoma virus mutant and do not have kinase activity. The transformation associated proteins may represent rat cellular factors activated during the transformation of rat cells by Moloney murine sarcoma virus.     

Heat shock proteins within the mammalian cell cycle: relationship to thermal sensitivity, thermal tolerance, and cell cycle progression

We have measured endogenous and induced rates of 70-kD, 89-kD, and 110-kD heat shock proteins in highly pure G1-, S-, or G2-M phase fractions of Chinese hamster fibroblasts (CHO) separated by fluorescence-activated cell sorting (FACS). Relative rates of synthesis of all three polypeptides as measured by two-dimensional gel electrophoresis were similar throughout the cell cycle, and therefore, endogenous levels were unlikely to explain the thermal sensitivity of S-phase cells. Distinct heterogeneity in induced rates of these polypeptides was noted in all phase fractions. Enhanced rates of 70-kD polypeptide were measured in S and G2-M as compared to G1 following heat shock. Little increase in either the 89-kD or 110k-kD heat shock proteins was observed in heated G1 cells. This heterogeneity in induced rates of synthesis was in contrast to the similarity in thermal tolerance expression kinetics between each phase. Finally, enhanced synthesis of these polypeptides appeared unrelated to regulation of either heat-induced cell cycle delay or to the resumption of phase-specific progression after heat shock as measured by simultaneous flow cytometric measurement of incorporated BrdUrd and DNA content.     

Unique sequence, ski, in Sloan-Kettering avian retroviruses with properties of a new cell-derived oncogene.

The Sloan-Kettering viruses (SKVs) are a group of transforming retroviruses that were isolated from chicken embryo cells which had been infected with the avian leukosis virus transformation-defective Bratislava 77 (tdB77). Each of the SKV isolates was shown to contain multiple genomes of different sizes indicating the presence of several viruses in addition to tdB77. To identify and characterize the putative transforming gene(s) of the SKVs, we used hybridization selection to isolate the fraction of a representative cDNA which was SKV specific. Both solution and blot hybridization studies with viral RNAs showed that the specific probe contained a sequence, ski, that was at least partially held in common by the multiple SKV genomes. This conclusion was confirmed by the observation that a molecularly cloned ski probe also hybridized to each of the multiple SKV genomes. Southern blots of chicken DNA revealed homologs of ski (c-ski) which were not associated with endogenous viral loci. Results showing that c-ski was expressed in polyadenylated cytoplasmic RNA of uninfected chicken cells indicated that it is a functional gene. Other data showed that c-ski was conserved in avian and mammalian evolution, suggesting a functional role for the gene in species other than chickens. Using ski cDNA in solution hybridizations with viral RNAs and in Southern blot hybridization with cloned retroviral oncogenes, we did not detect any relationship between ski and any of 15 previously identified oncogenes.