Spontaneous Variants Research Articles

Extracellular matrix-modulated expression of human cell surface glycoproteins A42 and J143. Intrinsic and extrinsic signals determine antigenic phenotype.

We have used serologic, biochemical, and genetic methods to characterize two stage-specific human differentiation antigens of neural and melanocytic cells: A42 (57,000 Mr glycoprotein) and J143 (140,000/30,000 Mr glycoprotein). The genes determining A42 and J143 cell surface expression in rodent-human hybrids were chromosomally mapped, and the respective human chromosomes were introduced into rodent cells derived from distinct differentiation lineages. Serologic analysis of the resulting hybrid clones has permitted the identification of two types of regulatory signals determining A42 and J143 expression. First, both antigens are expressed in hybrids constructed with antigen-positive human cells and also in certain hybrids constructed with antigen-negative human cells, indicating that intrinsic signals provided by the differentiation program of the rodent fusion partner induce antigen expression. Second, a series of human-mouse neuroblastoma hybrids, which are A42- or J143- when cultured on plastic surfaces, can be induced to express the antigens when cultured on substrates coated with extracellular matrix (ECM) produced by bovine corneal endothelial cells or fibronectin. This induction of antigen expression by extrinsic, ECM-derived signals is accompanied in the neuroblastoma hybrids by increased substrate adhesiveness and cell spreading and by characteristic changes in cell morphology. A similar program of phenotypic changes is also seen in spontaneous variants of human neuroblastoma and Ewing's sarcoma cells and in ECM-induced Ewing's sarcoma cells. These findings suggest that ECM-derived signals have a role analogous to mitogens and soluble differentiation factors in modulating differentiation phenotypes and tissue-specific patterns of cell surface antigen expression.

Overlapping deletion in two spontaneous phase variants of Coxiella burnetii.

Chromosomal DNA from the Nine Mile phase I strain of Coxiella burnetii (CB9MIC7) was cloned into the cosmid vector pHC79. The resulting gene library was probed with a radiolabelled HaeIII fragment present in the parental strain but absent from a spontaneously derived Nine Mile phase II strain (CB9MIIC4). The insert, which includes the missing HaeIII fragment, was 38.5 kb in length. When DNA from this cosmid clone was hybridized to genomic DNA of the parental CB9MIC7 and its derivative CB9MIIC4, a number of fragments were missing or altered in the latter strain. Restriction mapping localized the fragments to a contiguous portion of the chromosomal DNA fragment. The data were consistent with an 18 kb deletion in the chromosome of CB9MIIC4. Another intrastrain spontaneous derivative, CB9MI514, also lacked the sentinel HaeIII fragment and carried a deletion of approximately 29 kb within the same cloned insert. Both deletions appeared to share a common terminus, within the limits of resolution. In all other strains investigated, both phase I and phase II, the DNA represented by the insert seemed intact. The strains examined were representative of various stages of phase variation. The relationship between the observed deletions and the mechanism of phase transition in Nine Mile strains is discussed.

Isolation of mycoplasma virus L2 insertion variants and miniviruses.

We isolated two spontaneous variants of mycoplasma virus L2. Both variants, designated L2ins1 and L2ins2, contained a 3.1-kilobase-pair (kbp) insertion in the 11.8-kbp wild-type L2 genome. The insert DNA was shown to be derived from two noncontiguous regions of the L2 genome, and L2ins1 and L2ins2 differed only in the location of the 3.1-kbp insertion. We also isolated L2 miniviruses from serial passages of L2, L2ins1, and L2ins2 viruses. Miniviruses contained circular DNA molecules of 3.1 kbp or multimers of 3.1 kbp. Minivirus 3.1-kbp DNAs had the same sequences as the 3.1-kbp insert DNAs found in L2ins1 and L2ins2 viruses. Miniviruses were not infectious and interfered with the growth of L2, L2ins1, and L2ins2 viruses; hence, L2 miniviruses appeared to be defective interfering particles.

Structure and transforming function of transduced mutant alleles of the chicken c-myc gene.

A small retroviral vector carrying an oncogenic myc allele was isolated as a spontaneous variant (MH2E21) of avian oncovirus MH2. The MH2E21 genome, measuring only 2.3 kilobases, can be replicated like larger retroviral genomes and hence contains all cis-acting sequence elements essential for encapsidation and reverse transcription of retroviral RNA or for integration and transcription of proviral DNA. The MH2E21 genome contains 5' and 3' noncoding retroviral vector elements and a coding region comprising the first six codons of the viral gag gene and 417 v-myc codons. The gag-myc junction corresponds precisely to the presumed splice junction on subgenomic MH2 v-myc mRNA, the possible origin of MH2E21. Among the v-myc codons, the first 5 are derived from the noncoding 5' terminus of the second c-myc exon, and 412 codons correspond to the c-myc coding region. The predicted sequence of the MH2E21 protein product differs from that of the chicken c-myc protein by 11 additional amino-terminal residues and by 25 amino acid substitutions and a deletion of 4 residues within the shared domains. To investigate the functional significance of these structural changes, the MH2E21 genome was modified in vitro. The gag translational initiation codon was inactivated by oligonucleotide-directed mutagenesis. Furthermore, all but two of the missense mutations were reverted, and the deleted sequences were restored by replacing most of the MH2E21 v-myc allele by the corresponding segment of the CMII v-myc allele which is isogenic to c-myc in that region. The remaining two mutations have not been found in the v-myc alleles of avian oncoviruses MC29, CMII, and OK10. Like MH2 and MH2E21, modified MH2E21 (MH2E21m1c1) transforms avian embryo cells. Like c-myc, it encodes a 416-amino-acid protein initiated at the myc translational initiation codon. We conclude that neither major structural changes, such as in-frame fusion with virion genes or internal deletions, nor specific, if any, missense mutations of the c-myc coding region are necessary for activation of the basic oncogenic function of transduced myc alleles.

Cyclic AMP-dependent protein kinase promotes glucocorticoid receptor function.

Murine lymphoma cell lines such as WEHI-7 exhibit a cytolytic response to both cAMP and glucocorticoids. We have exploited this behavior to ask if cyclic AMP-dependent protein kinase plays a role in regulating glucocorticoid receptor function. We have found that cAMP-resistant cell lines containing a defective cAMP-dependent protein kinase activity give rise to spontaneous steroid-resistant variants at a high frequency (approximately 10(-7)) relative to wild type cells (less than 10(-10)). Unlike previous results with wild type cells, nearly complete loss of glucocorticoid receptor function was observed in a single selection using unmutagenized cAMPr derivatives of WEHI-7. Thus, the initial selection of the cAMPr phenotype serves as a permissive step toward the acquisition of glucocorticoid resistance in WEHI-7. In addition, cAMP was found to increase the levels of steroid binding in these cell lines, and the dose response was dependent upon the phenotype of the cyclic AMP-dependent protein kinase. The results demonstrate an important role for cAMP in regulating glucocorticoid receptor activity and strongly suggest that this novel two-step selection scheme leads to the isolation of new forms of glucocorticoid resistance.

Construction of a new vector for the expression of foreign genes inZymomonas mobilis

Broad host range plasmids have previously been shown to be suitable as vectors to introduce antibiotic resistance genes intoZ. mobilis. However, attempts to use these vectors to carry other genes with enteric promoters and controlling elements have resulted in limited success due to poor expression. Thus we have constructed a promoter cloning vector in a modified pBR327 and used this vector to isolated 12 promoters fromZ. mobilis which express various levels of β-galactosidase inEscherichia coli. Four of these were then subcloned into pCVD 305 for introduction intoZ. mobilis. All expressed β-galactosidase inZ. mobilis with activities of 100 to 1800 Miller units. One of these retained aBamHl site into which new genes can be readily inserted immediately downstream from theZ. mobilis promoter. Genetic traits carried by pCVD 305 were initially unstable but spontaneous variants were produced during sub-culture in which the plasmid was resistant to curing at elevated temperature. One of these variants was examined in some detail. The increased stability of this variant appears to result from an alteration in the plasmid rather than a chromosomal mutation or from chromosomal integration.

Vigor and variation expressed by anther-derived doubled haploids of burley tobacco (Nicotiana tabacum L.). II. Evaluation of first- and second-cycle doubled haploids

First- and second-cycle doubled haploids of burley tobacco (Nicotiana tabacum L.) were examined to ascertain the significance of source genotype on the performance of anther-derived material and to determine the magnitude of changes from the haploid-production and chromosome-doubling stages. The anther-derived lines evaluated in this study did not exhibit siginficant yield reductions. However, significant interactions between the cycle comparison and the source genotypes indicated that source genotype can affect the degree of vigor reduction observed. Significant variation was also observed among the second-cycle doubled haploids examined in this study. Partitioning of this variation indicates that most of it is due to alteration present at the haploid-production step instead of the chromosome-doubling step. In general, the variation present in the second-cycle doubled haploids was distributed around the values observed for the source first-cycle doubled haploids. Even though unexpected variation is often observed in anther-derived materials, these results suggest that reduced vigor is not intrinsically associated with androgenetic lines of burley tobacco. The most plausible explanations for the variation and vigor losses in anther-derived lines propose that natural phenomena, given the opportunity for expression via anther culture, are responsible. Spontaneous variants present in the gametic pool of inbreds or first-cycle doubled haploids offer one possible explanation for the performance observed in anther-derived lines of tobacco.

Distinct T-cell proliferative responses to 13762A rat mammary adenocarcinoma and derived clones

We examined the in vitro responses of immune lymphocytes to the tumor antigens of the syngeneic rat mammary adenocarcinoma 13762A. This tumor readily metastasizes to lymph node and lungs and is poorly immunogenic. Rats were immunized with a highly immunogenic clone (18A) which was isolated as a spontaneous variant from the parental 13762A tumor. Clone 18A grew progressively in irradiated rats but regressed completely in normal rats. Animals immune to 18A tumor were also immune to parental 13762A. Lymphocytes obtained from the spleen and peritoneum of immune rats were tested for specific proliferation to parental 13762A tumor and clone 18A to determine whether similar cross-reactivity to these tumors occurred in vitro. We found an anatomical difference in localization of immune lymphocytes which reacted to the two tumor cell lines. Immune peritoneal exudate cells (PEC) responded strongly to clone 18A but poorly to 13762A, while immune spleen cells from the same animals responded predominantly to 13762A tumor. After 7 days culture, PEC proliferating in response to clone 18A contained 84–95% W3/25 + T-helper cells, and only 5–8% OX8 + cytotoxic/suppressor cells, while analogous cultures of spleen cells responding to parental 13762A tumor consisted of 60–80% W3/25 + cells and 20–23% OX8 + cells. Immune spleen cell cultures stimulated with 13762A tumor generated cytotoxic lymphocytes which specifically lysed both parental 13762A and clone 18A cells. We conclude that despite cross-reactivity in vivo and in vitro, antigens present on 13762A and 18A tumor cells stimulated different subsets of immune T cells.

987P fimbriae from porcine enterotoxigenicEscherichia coli: Characterization, N-terminal amino acid sequence and immunization with purified antigen

The 987P fimbrial antigen was purified from a spontaneous overproducing variant. The protein was characterized with respect to Mr, amino acid sequence and partial covalent structure. The purified protein was used for vaccination trials, and excellent protection of piglets upon challenge with 987P positive enterotoxigenic strains was seen.

An antigenic analysis using monoclonal antibodies of influenza A (H3N2) viruses isolated from an epidemic in a semi-closed community.

Seventy-eight influenza A (H3N2) viruses isolated from a single epidemic in a semi-closed community involving 203 clinical cases were characterized using a panel of monoclonal antibodies to virus haemagglutinin (HA). Thirty groups of antigenically distinguishable viruses were detected but the majority (41%) of the viruses belonged to two serological groupings, designated 11 and 17. Viruses in serological group 11 were present throughout the outbreak. The greatest diversity of antigenic variants occurred at the time of the epidemic peak. Antigenic differences among the HAs of the viruses were also detected using polyclonal human and animal antisera. The electrophoretic mobility of virus-induced structural and non-structural polypeptides and of the RNA of viruses of representative serological groups was similar or identical, suggesting that new introductions of viruses did not occur during the progress of the epidemic. The evolution of influenza A (H3N2) epidemics even in small communities appears to be complex, although a contribution to the observed antigenic microheterogeneity of the HA by spontaneous variants arising in the laboratory cannot be excluded.

Nucleotide sequence of an insertion sequence (IS) element identified in the T-DNA region of a spontaneous variant of the Ti-plasmid pTiT37.

We have identified and determined the nucleotide sequence of an IS element (IS136) of Agrobacterium tumefaciens. This is the first IS element isolated and sequenced from a nopaline type Ti-plasmid. Our IS element has 32/30 bp inverted repeats with 6 mismatches, is 1,313 bp long and generates 9 bp direct repeats upon integration. IS136 has 3 main open reading frames (ORF's). Only ORF1 (159 codons) is preceded by sequences that are proposed to serve functional roles in transcriptional and translational initiation. No DNA sequence homology was found between IS136 and IS66, an IS element isolated from an octopine type Ti-plasmid.

The possession of three novel coli surface antigens by enterotoxigenic Escherichia coli strains positive for the putative colonization factor PCF8775.

A possible colonization factor, E8775, has previously been described for enterotoxigenic Escherichia coli strains of serogroups O25, O115 and O167. Re-examination of these strains by immunodiffusion has revealed that the antigenic nature of this factor, renamed putative colonization factor (PCF) 8775, is more complex than was first thought. All the strains of serogroup O25 tested possessed two antigenic components, termed CS4 and CS6, and gave mannose-resistant haemagglutination (MRHA) of human and bovine erythrocytes. Spontaneous variants possessing CS6 only did not give MRHA. Strains of serogroups O115 and O167 had the antigenic components CS5 and CS6, and gave MRHA of human, bovine and guinea-pig erythrocytes. Using immune electron microscopy, the components CS4 and CS5 were identified as fimbriae. No fimbriae were associated with CS6.

Ventricular ectopic activity with spontaneous variant angina: Frequency and relation to transient ST segment deviation

This study was performed (1) to assess the frequency of ventricular ectopic activity (ventricular bigeminy, couplets, or ventricular tachycardia) during spontaneous variant angina; (2) to assess the relation between ventricular ectopy and the severity and duration of ischemia; and (3) to evaluate the precise temporal relation between episodes of ischemia and ventricular ectopy. Fifteen ambulatory patients with variant angina (12 men, 3 women, aged 50 ± 8 [mean ± SD] years) had Holter monitoring for 24 hours/week for 10 months (total, 10,238 hours of monitoring), from which the following were measured during each episode of ST deviation (elevation or depression): duration of ST deviation (minutes), maximal ST deviation (millivolts), presence of ventricular ectopic activity, and timing of ventricular ectopy in relation to ST deviation. Of 645 eplsodes of ST deviation, 79 (12.2%) had associated ectopy, almost all of which occurred in three patients. The 79 episodes of ST deviation with ectopy lasted 4.6 ± 3.3 minutes and averaged 0.16 ± 0.12 mV, whereas the 566 episodes of ST deviation without ectopy lasted 4.7 ± 6.1 minutes and averaged 0.17 ± 0.11 mV (NS in comparison to the 79 episodes with ectopy). Of 489 episodes of ST elevation, 72 (14.7%) were accompanied by ventricular ectopy; of 156 episodes of ST depression, only seven (4.5%) had ectopy ( χ 2 = 11.531, p < 0.001). Of the 79 episodes of ventricular ectopy, almost all appeared during a period of increasing or maximal ST deviation, whereas only two appeared as ST deviation was resolving. Thus, in ambulatory patients with variant angina, (1) ventricular ectopy occurs only in an occasional individual and in association with only about 10% to 20% of episodes of transient ST deviation; (2) it occurs more often with transmural (ST elevation) than with subendocardial (ST depression) ischemia, but there is no clear relation between ectopy and the duration or magnitude of ST deviation; and (3) ectopy almost always appears during the initial 2 to 3 minutes of ST deviation, at a time when its magnitude is increasing or maximal.

Characterization and infectivity of a spontaneous variant isolated fromFrankia sp. WEY 0131391

A spontaneous variant, obtained from aFrankia isolate fromAlnus rubra nodules, was compared with the parent strain with regard to infectivity, nitrogenase activity, and electrophoretic and immunological profiles. Both the parent and the variant strain were equally effective in inducing nodulation in seedlings ofA. rubra. All inoculated plants had an active nitrogenase system as measured by the acetylene reduction assay. Electrophoresis of whole cell homogenates on SDS-polyacrylamide slab gels showed similar electrophoretic profiles; however, the variant strain also exhibited striking differences in protein patterns that distinguish it from the parent strain. Immunological analysis of the originalFrankia strain and its variant revealed shared antigens as well as immunologically distinct antigenic determinants in the two strains. The variant strain exhibits a distinct morphology and growth patterns which remain stable after many passages through culture.

Ultraviolet-irradiation induced and spontaneous mutation of Rhizobium trifolii 11B in relation to water-soluble and water-insoluble polysaccharide production ability

Rhizobium trifolii 11B was u.v. irradiated and nine u.v. mutants have been isolated. Among the mutants, only one, R. trifolii 21M11B, produced more (752 mg/100 ml) water-soluble polysaccharide than the parent (704 mg/100 ml). The composition of water-soluble polysaccharide from u.v. mutants differed from that of the parent, R. trifolii 11B, and none of its u.v. mutants produced water-insoluble polysaccharide as detected by the Aniline Blue method. Storage of u.v. mutants for 2 months at 5°C gave four spontaneous variants which acquired the ability to produce water-insoluble polysaccharide. The spontaneous mutants also retained their water-soluble polysaccharide producing ability. The water-soluble polysaccharide produced by these mutants was characterized as curdlan type. The chemistry of water-soluble and water-insoluble polysaccharides was also ascertained.

Loss of sulfide adaptation ability in a thermophilic Oscillatoria

A spontaneous variant incapable of anoxygenic photosynthesis was derived from a fully competent strain of Oscillatoria amphigramulata which was originally isolated from a high sulfide-containing hot spring of New Zealand. Although the variant (Oa-2) acquired a slight ability to photosynthesize in the presence of 0.3–0.4 mM sulfide, this was only after a 24 h exposure to sulfide and represented oxygenic photosynthesis only. Unlike the parent strain, the incompetent variant never grew in the presence of sulfide >0.05 mM, nor was there any relief of the inhibition by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] of CO2 photoincorporation when sulfide was present. The variant strain has retained all of these characteristics over a 4 year period with monthyl transfers in non-sulfide medium. The wild type, under identical conditions, has retained all of its competence with respect to sulfide.

Spontaneous phase variation in Bordetella pertussis is a multistep non-random process.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non-pathogenic upon culturing in vitro. Spontaneous variants of the Tohama and #165 pathogenic strains of B. pertussis were selected by their ability to grow on synthetic and semi-synthetic solid media. The frequency of these variants was between 10(-6) and 10(-7). About 250 variant strains were screened for the presence of virulence-associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7-11% of the variants displayed all traits, 17% of the variants carried the toxin and FHA, 5-11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin quantitatively affected the other traits. These results suggest that phase variation in B. pertussis is a non-random process, involving multistep disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. In contrast, all 300 variants of strain #18323 of B. pertussis, which were able to grow on the selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral growth capability.

Isolation of sodium chloride-tolerant callus line of Cicer arietinum L. cv. BG-203

A NaCl-tolerant callus line of Cicer arietinum has been isolated, as a spontaneous variant, on agar-solidified MS-medium supplemented with 100 mM NaCl. The growth of this line, in the presence of 100 mM NaCl, was comparable to that of the NaCl-sensitive callus line growing in the absence of NaCl. Regarding relative tolerance of the two callus lines towards NaCl (0 to 200 mM), the tolerant line performed poorly in the absence of NaCl and exhibited optimal growth at 50 mM NaCl. The tolerance persisted even after three passages of 4-wk each, tested so far, away from the selective agent.

Isolation and characterization of Bordetella pertussis phenotype variants capable of growing on nutrient agar: comparison with phases III and IV.

Unsupplemented nutrient agar (NA) was used to select spontaneous phenotype variants (PVs) of Bordetella pertussis Tohama I and 3779 which, by their growth on NA, could possibly be considered equivalent to phase IV in the system of Leslie and Gardner (P.H. Leslie and A.D. Gardner, J. Hyg. 31:423-434, 1931) or phase III in the system of Kasuga et al. (T. Kasuga, Y. Nakase, K. Ukishima, and K. Takatsu, Kitasato Arch. Exp. Med. 26:121-134, 1953). NA growers (Gna+) were selected from the flat, nonhemolytic, non-NA grower (Dom- Hly- Gna-) PV of both strains at a rate of between 10(-7) to 10(-8) per cell per generation. When cultured on Bordet-Gengou agar (BGA), more than one colony type was observed in strain 3779; these all retained the Gna+ characteristic during 10 to 30 passages on BGA. Analysis of 125I-surface-labeled whole cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no major changes between the Dom- Hly- Gna+ PV and their Dom- Hly- Gna- PV parents in polypeptide profile (by Coomassie stain), in surface exposure of proteins (by autoradiography), or in lipopolysaccharide profile (by silver stain). Increased resistance to oleic acid, tetracycline, erythromycin, rifampin, and penicillin G, however, was characteristic for the Dom- Hly- Gna+ PV. Five phase IV strains and a phase III B. pertussis strain had similar antibiotic and oleic acid sensitivity profiles as the Dom- Hly- Gna+ isolates and plated with similar efficiency on NA, despite heterogeneity in BGA colonial morphology and lipopolysaccharide profile.

Ventricular arrhythmias during ergonovine-induced episodes of variant angina

Of 95 consecutive patients with active variant angina who underwent ergonovine testing in the coronary care unit while off treatment, 24 (25%) developed serious ventricular arrhythmias: ventricular tachycardia in eight, bigeminy in seven, pairs in five, and frequent ventricular extrasystoles in four. Ergonovine-induced arrhythmias were observed more often in patients with anterior than inferior ST segment elevation ( p < 0.05). ST segment elevation was significantly higher (10.3 ± 8.1 vs 3.1 ± 2.1 mm) in patients who developed arrhythmias. All ventricular arrhythmias began within 3 minutes after the onset of ST segment elevation. The intravenous administration of nitroglycerin eliminated arrhythmias in 22 of 24 cases; in only two patients did ventricular arrhythmias develop after the administration of nitroglycerin. Serious ventricular arrhythmias were found during spontaneous variant angina attacks in 14 of 24 patients with ergonovine-induced arrhythmias compared to 16 of 71 patients without ergonovine-induced arrhythmias ( p < 0.001). We conclude that arrhythmias during ergonovine testing are most often caused by ischemia and not reperfusion. Patients with arrhythmias during ergonovine-induced attacks are more likely to have arrhythmias during spontaneous attacks.