Showing papers in "Molecular Genetics and Genomics in 1977"
TL;DR: Derivatives of phage λ are described for use as vectors for fragments of DNA generated with the HindIII and EcoRI restriction enzymes to permit the ready distinction between recombinant and vector phages by the colour of the plaques.
Abstract: Derivatives of phage lambda are described for use as vectors for fragments of DNA generated with the HindIII and EcoRI restriction enzymes. With some vectors, hybrid molecules are recognised by a change from a turbid to a clear plaque morphology resulting from the insertion of a fragment of DNA into the lambda gene coding for the phage regressor. Other vectors contain a central, replaceable fragment of DNA which imparts a readily recognisable phenotype. This central fragment may include either a gene for a mutant transfer RNA (suppressor) or a part of the lacZ gene of E. coli able to complement a lacZ host. The appropriate lacZ host and indicator plates permit the ready distinction between recombinant and vector phages by the colour of the plaques.
675 citations
TL;DR: The mutation diminishes UV mutagenesis and UV reactivation of phages λ without affecting the inducibility of phophage λ nor the inhibition of cell division following UV irradiation.
Abstract: Mutants of E. coli defective in susceptibility to UV-induction of mutations were isolated by direct screening for their UV nonmutable phenotype (Umu-). Screening of about 30,000 mutagenized clones of a uvr-B derivative of AB1157 yielded six Umu- strains. The mutants can be classified into three groups by the location of the mutations, umuA, umuB and umuC. Mutations umuA and umuB are, respectively, mapped close to lexA and recA genes and mutations at both loci partially reduce UV mutagenesis. The locus of umuC is between hemA and purB and the mutations at this new locus result in a moderate increase of UV sensitivity. The mutation diminishes UV mutagenesis and UV reactivation of phage lambda without affecting the inducibility of phophage lambda nor the inhibition of cell division following UV irradiation. Related properties of an isogenic strain of a recF- mutant are compared with those of umuC-.
546 citations
TL;DR: Protoplasts of soybean and N. glauca were induced to fuse with polyethylene glycol (PEG 1540) and up to 39% of the protoplasts in the treated population were heterokaryocytes, which indicated a tendency to stick together and break into pieces in the later cell generations.
Abstract: Protoplasts of soybean and N. glauca were induced to fuse with polyethylene glycol (PEG 1540). Up to 39% of the protoplasts in the treated population were heterokaryocytes. When the heterokaryocytes were isolated and individually cultivated they divided indefinitely and each produced many millions of cells within 2–3 months.
470 citations
TL;DR: A diploid strain has been obtained by aposporus regeneration from a hybrid sporophyte and the phenotypes of progeny resulting from the self-fertilisation of this strain have been analysed.
Abstract: Eighteen nutritional mutants have been isolated in the haploid, monoecious moss, Physcomitrella patens: five nicotinic acid auxotrophs, four p-aminobenzoic acid auxotrophs, four adenine auxotrophs, two amino acid requiring mutants and three nitrate non-utilising mutants. Seventeen of them were obtained using total isolation; one was isolated selectively. Strains resistant to the amino acid analogues, D-serine and p-fluorophenyl-alanine, and the purine analogue, 8-azaguanine, have been selected. Many of the auxotrophs are self-sterile. Crosses between auxotrophic strains have been effected and the progeny analysed. No linkage has been detected. Nicotinic acid auxotrophy has resulted from mutation in at least two genes. Self-sterility segregates as a pleiotropic effect of four mutations which produce nutritional dependence. A diploid strain has been obtained by aposporus regeneration from a hybrid sporophyte and the phenotypes of progeny resulting from the self-fertilisation of this strain have been analysed.
400 citations
TL;DR: Insertion of the transposable genetic element Tn1 into different sites of plasmid ColE1 results in a number of mutant phenotypes, and non-transmissibility is correlated with reduced relaxation complex.
Abstract: Insertion of the transposable genetic element Tn1 into different sites of plasmid ColE1 results in a number of mutnat phenotypes. Whereas all plasmid examined were present in normal amount, all showed reduced immunity to killing by colicin E1. Of six insertions isolated after conjugation, five fail to produce colicin, are conjugally proficient (transmissible), and map within a 500 nucleotide region of the genome. The other is conjugally deficient, produces colicin normally and maps close to two others with a similar phenotype isolated after transformation. Of four others isolated after transformation, two have similar properties to the original five transmissible plasmids. The other two are nontransmissible and produce colicin. Non-transmissibility is correlated with reduced relaxation complex. Patterns of protein synthesis in minicells by ColE1 and ColE1 :: Tn1 plasmids have been examined: all ColE1 plasmids containing Tn1 show an altered pattern of ColE1 protein synthesis in addition to three presumptive Tn1-specified proteins, one of which is shown to be beta-lactamase. ColE1 :: Tn1 plasmids can be inserted into the conjugative plasmid R64drd11 to form a cointegrate in which ColE1 and Tn1 function can be expressed.
263 citations
TL;DR: Results confirmed that the 18×106 dalton plasmid is not, and does not include, SCP1, which has not so far been isolated by any of a variety of methods, and served to identify a second sex factor, SCP2, postulated to be present in SCP2+ state in the starting strains and to be capable of mutation to a variant form,SCP2*, with enhanced sex factor activity.
Abstract: Covalently closed circular (ccc) DNA of uniform monomer size (c. 18×106 daltons) and restriction endonuclease cleavage pattern was isolated from strains of S. coelicolor A3(2) of differing constitution in respect of the SCP1 sex factor: SCP1+, SCP1′, SCP1- and NF (integrated SCP1). No such ccc DNA was found in strains of S. lividans 66 or S. parvulus ATCC 12434 whether or not they contained SCP1. These results confirmed that the 18×106 dalton plasmid is not, and does not include, SCP1, which has not so far been isolated by any of a variety of methods.
233 citations
TL;DR: An examination of the cross-reactivity of this response with other mutagens has shown that it is a generalised mechanism affecting alkylation damage to DNA, which cannot, however, be induced by UV or the UV-mimetic mutagen, 4-nitroquinoline 1-oxide, nor act on lesions put into DNA by thosemutagens.
Abstract: We have described previously an inducible response in Escherichia coli which occurs during growth on low levels of the methylating agent, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and which enables cells both to survive better and to be less mutated by a subsequent challenge dose of MNNG than control cultures (Samson and Cairns, 1977). We show here that this response is distinct from previously characterised pathways of DNA repair, and particularly from the SOS response, which is another inducible effect resulting from DNA damage. An examination of the cross-reactivity of this response with other mutagens has shown that it is a generalised mechanism affecting alkylation damage to DNA. It cannot, however, be induced by UV or the UV-mimetic mutagen, 4-nitroquinoline 1-oxide, nor act on lesions put into DNA by those mutagens.
207 citations
TL;DR: Mutants with defective carbon catabolite repression have been isolated in the yeast Saccharomyces cerevisiae using a selective procedure based on the fact that invertase is a glucose repressible cell wall enzyme which slowly hydrolyses raffinose to yield fructose and that the inhibitory effects of 2-deoxyglucose can be counteracted by fructose.
Abstract: Mutants with defective carbon catabolite repression have been isolated in the yeast Saccharomyces cerevisiae using a selective procedure. This was based on the fact that invertase is a glucose repressible cell wall enzyme which slowly hydrolyses raffinose to yield fructose and that the inhibitory effects of 2-deoxyglucose can be counteracted by fructose. Repressed cells were plated on a raffinose--2-doexyglucose medium and the resistant cells growing up into colonies were tested for glucose non-repressible invertase and maltase. The yield of regulatory mutants was very high. All were equally derepressed for invertase and maltase, no mutants were obtained with only non-repressible invertase synthesis which was the selected function. A total of 61 mutants isolated in different strains were allele tested and could be attributed to three genes. They were all recessive. Mutants in one gene had reduced hexokinase activities, the other class, located in a centromere linked gene, had elevated hexokinase levels and was inhibited by maltose. Mutants in a third gene were isolated on a 2-deoxyglucose galactose medium and had normal hexokinase levels. A partial derepression was observed for malate dehydrogenase in all mutants. Isocitrate lyase, however, was still fully repressible.
195 citations
TL;DR: N-Acetylglutamate synthase, the enzyme which replenishes the cycle of acetylated intermediates in ornithine synthesis of Pseudomonas, appears to be essential for arginine synthesis since argA mutants showed no growth on unsupplemented minimal medium.
Abstract: Six loci coding for arginine biosynthetic enzymes in Pseudomonas aeruginosa strain PAO were identified by enzyme assay: argA (N-acetylglutamate synthase), argB (N-acetylglutamate 5-phosphotransferase), argC (N-acetylglutamate 5-semialdehyde dehydrogenase), argF (anabolic ornithine carbamoyl-transferase), argG (argininosuccinate synthetase), and argH (argininosuccinase). One-step mutants which had a requirement for arginine and uracil were defective in carbamoylphosphate synthase, specified by a locus designated car. To map these mutations we used the sex factor FP2 in an improved interrupted mating technique as well as the generalized transducing phages F116L and G101. We confirmed earlier studies, and found no clustering of arg and car loci. However, argA, argH, and argB were mapped on a short chromosome segment (approx. 3 min long), and argF and argG were cotransducible, but not contiguous. N-Acetylglutamate synthase, the enzyme which replenishes the cycle of acetylated intermediates in ornithine synthesis of Pseudomonas, appears to be essential for arginine synthesis since argA mutants showed no growth on unsupplemented minimal medium.
181 citations
TL;DR: Some of a set of independently arising Tol- (non toluate-utilising) derivatives of Pseudomonas putida mt-2 have lost the unique plasmid present in the parent strain.
Abstract: Some of a set of independently arising Tol- (non toluate-utilising) derivatives of Pseudomonas putida mt-2 have lost the unique plasmid present in the parent strain. In others this plasmid has suffered a deletion of a specific region of about 27 Md.
173 citations
TL;DR: Cytological and haploidization examinations showed that the fusion cells are not heterokaryons, and that the increased amount of genetic material is situated in one nucleus.
Abstract: Protoplasts of methionine-and lysine-requiring h- mutants isolated from the L972 h- strain of Schizosaccharomyces pombe were fused. The protoplasts were obtained from the cells with enzymes produced by Trichoderma viride. When a mixture of the protoplasts was treated with 30% PEG 4000 solution containing 10 mM CaCl2, cell fusion and complementation was attained with a frequency of 0.17%. Both fusion partners were recovered among the spores after crossing of the fusion products with the strain M210 ade6 h+. Cytological and haploidization examinations showed that the fusion cells are not heterokaryons, and that the increased amount of genetic material is situated in one nucleus.
TL;DR: The observations suggest that the drug resistance plasmid pKM101 does not exert its effects by altering the regulation of the cell's error-prone repair system but rather by supplying a mechanistic component or components.
Abstract: The drug resistance plasmid pKM101 plays a mojor role in the Ames Salmonella/microsome carcinogen detecting system by enhancing chemical mutagenesis. It is shown that in Escherichia coli K-12 the plasmid pKM101 enhances both spontaneous and methyl methanesulfonate-caused reversion of an ochre mutation, bacterial survival after ultraviolet irradiation, and reactivation of ultraviolet-irradiated lambda in unirradiated cells. All these effects are shown to be dependent on the recA+ lexA+ genotype but not on the recB+ recC+ or recF+ genotypes. The recA lexA-dependence of the plasmid-mediated repair and mutagenesis suggests an interaction with the cell's inducible error-prone repair system. The presence of pKM101 is shown to cause an additional increase in methyl methanesulfonate mutagenesis in a tif mutant beyond that caused by growth at 42 degrees. The presence of the plasmid raises the level of the Weigle-reactivation curve for the raactivation of ultraviolet-irradiated lambda in E. coli and causes a shif of the maximum to a higher UV fluence. These observations suggest that pKM101 does not exert its effects by altering the regulation of the cell's error-prone repair system but rather by supplying a mechanistic component or components.
TL;DR: A cleavage map of bacteriophage P1 DNA was established by reciprocal double digestion with various restriction endonucleases by determining the relative order of the PstI, HindIII and BglII sites, as well as the order of 13 out of the 14 BamHI sites and of 17 of the 26 EcoRI sites was determined.
Abstract: A cleavage map of bacteriophage P1 DNA was established by reciprocal double digestion with various restriction endonucleases. The enzymes used and, in parenthesis, the number of their cleavage sites on the P1clts genome are: PstI (1), HindIII (3), BglII (11), BamHI (14) and EcoRI (26). The relative order of the PstI, HindIII and BglII sites, as well as the order of 13 out of the 14 BamHI sites and of 17 out of the 26 EcoRI sites was determined. The P1 genome was divided into 100 map units and the PstI site was arbitrarily chosen as reference point at map unit 20.
TL;DR: A bacterial mutation affecting λ DNA replication, called groPC756, has been mapped between the thr and leu bacterial loci as mentioned in this paper, and it is shown that the mutation at the groPC locus also interferes with bacterial growth at 42°C.
Abstract: A bacterial mutation affecting λ DNA replication, called groPC756, has been mapped between the thr and leu bacterial loci. Most of the parental λ DNA does not undergo even one round of replication in this host. Lambda mutants, called π, which map in the λ P gene are able to overcome the inhibitory effect of the groPC756 mutation. It is shown that the mutation at the groPC locus also interferes with bacterial growth at 42°C. A λ-transducing phage, carrying the groPC+ allele, was isolated as a plaqueformer on groPC756 bacteria. Upon lysogenization, it restores both the gro
+ and temperature resistant phenotypes.
TL;DR: Transductional evidence that these strains carry nifB-A-F deletions andamination of the biochemical phenotype of several mutants suggests that the nifA product has a regulatory function, and nifK, nifD and nIFH are most probably the structural genes for nitrogenase.
Abstract: A series of mutants defective in nitrogen fixation (nif) were isolated in Klebsiella pneunoniae strain M5a1. The nif mutations were either located on plasmid pRD1 or on the K. pneumoniae chromosome. A total of 37 plasmid mutants and 28 chromosomal mutants were employed in complementation tests using the acetylene reduction technique. Most mutants could be assigned to one of seven nif cistrons: nifA, nifB, nifD, nifE, nifF, nifH, and nifK.
TL;DR: Dominance and complementation analyses have been carried out in partial diploids with mutations at the recA locus, and suggests that recA, zab and lexB may affect a common product, recA+.
Abstract: Dominance and complementation analyses have been carried out in partial diploids with mutations at the recA locus. These mutations modify in different ways the pleiotropic response normally exhibited by wild-type bacteria to an alteration in DNA replication (see Witkin, 1974; Radman, 1974); they either permit constitutive expression of this response at 42° (tif mutation: Castellazzi, George and Buttin, 1972a) or abolish it (recA and zab: Castellazzi, George and Buttin, 1972b; lexB: Blanco, Levine and Devoret, 1975).
TL;DR: Quantitative considerations on the porin-dependent permeability of the outer membrane show that there may be very large differences in the actual rates of penetration, even among the “permeable” substances.
Abstract: Four “pleiotropic transport” mutants of Escherichia coli B/r with decreased affinity for the uptake of most nutrients were found to lack a major outer membrane protein of 36,500 daltons (“porin”) previously shown to produce transmembrane diffusion channels in in vitro reconstitution experiments. Consequent decrease in outer membrane permeability was confirmed by measuring the transmembrane diffusion rate of 6-aminopenicillanic acid. Quantitative considerations on the porin-dependent permeability of the outer membrane show that (a) there may be very large differences in the actual rates of penetration, even among the “permeable” substances and (b) the numbers of porin molecules present in wild type cells is several orders of magnitude higher than that necessary for the uptake of rapidly diffusing substrates such as glocose from ordinary culture media. The absence of porin and the pleiotropic transport defect were always contransduced, and the mutation was mapped at 73.7 min between aroB and malT by P1 transduction. When “revertants” able to grow on low concentrations of lactose were selected, in addition to true revertants “suppressor” strains with increased amounts of non-porin membrane proteins were isolated.
TL;DR: A recessive mutant cat1-1, wild type CAT1, was isolated in Saccharomyces cerevisiae and was proposed that carbon catabolite repression acts in two ways: the direct way represses synthesis of sensitive enzymes, during growth on repressing carbon sources whereas the other way regulates the derepression process.
Abstract: A recessive mutant cat1-1, wild type CAT1, was isolated in Saccharomyces cerevisiae. It did not grow on glycerol nor ferment maltose even with fully constitutive, glucose resistant maltase synthesis. It prevented derepression of isocitrate lyase, fructose-1,6-diphosphatase and maltase in a constitutive but glucose sensitive maltase mutant. Derepression of malate dehydrogenase was retarded and slowed down. Sucrose fermentation and invertase synthesis was not affected. Respiration was normal. From this mutant, two reverse mutants were isolated. One was recessive, acted as a suppressor of cat1-1 and was called cat2-1, wild type CAT2; the other was dominant and allelic to CAT1 and designated CAT1-2d and cat2-1 caused an earlier derepression of enzymes studied but did not affect the repressed nor the fully derepressed enzyme levels. CAT1-2d and cat2-1 did not show any additive effects. It is proposed that carbon catabolite repression acts in two ways. The direct way represses synthesis of sensitive enzymes, during growth on repressing carbon sources whereas the other way regulates the derepression process. After alleviation of carbon catabolite repression, gene CAT1 becomes active and prevents the activity of CAT2 which functions as a repressor of sensitive enzyme synthesis. The CAT2 gene product has to be eliminated before derepression can actually occur. The time required for this causes a delay in derepression after the depletion of a repressible carbon source. cat1-1 cannot block CAT2 activity and therefore, derepression is blocked. cat2-1 is inactive and derepression can start after carbon catabolite repression has ceased. CAT1-2d permanently active as a repressor of CAT2 and eliminates the delay in derepression.
TL;DR: This paper constructed independent physical maps of the mtDNAs from three different wild-type Saccharomyces strains by double-digestion analysis and hybridization analysis, using restriction endonucleases EcoRI, HindII, HindIII, PstI, BamHI, Aval, HhaI, SalI and XhoI.
Abstract: 1)
We have constructed independent physical maps of the mtDNAs from three different wild-type Saccharomyces strains by double-digestion analysis and hybridization analysis, using restriction endonucleases EcoRI, HindII, HindIII, PstI, BamHI, Aval, HhaI, SalI and XhoI. Twentynine restriction enzyme sites have been localized on the mtDNA of Saccharomyces carlsbergensis, 47 on the mtDNA of Saccharomyces cerevisiae strain JS1-3D and 38 on the mtDNA of Saccharomyces cerevisiae strain KL14-4A.
2)
Although the three DNAs show considerable differences in their fragmentation patterns with most nucleases tested, the overall sequence organization of the three maps of 30–40 fragments is identical. Differences in the maps can be explained by extra restriction enzyme recognition sites, possibly located on inserted pieces of DNA and by insertions and deletions.
3)
Four major insertions (900, 1,500, 2,600 and 3,000 bp long) are found in KL14-4A mtDNA relative to Saccharomyces carlsbergensis mtDNA. These insertions are clustered in one quadrant of the mtDNA and account for the difference in size of these two mtDNAs (75,750 and 68,000 bp, respectively).
TL;DR: Somatic hybrids selected following treatment of mixtures of protoplasts from complementary auxotrophic strains with 50 mM CaCl2 at high pH have a morphology different from that of normal haploid strains, but similar to that of aposporously produced diploids.
Abstract: A technique has been developed for the isolation of large numbers of protoplasts from protonemal tissue of Physcomitrella patens, and for their regeneration to give whole plants. Somatic hybrids have been selected following treatment of mixtures of protoplasts from complementary auxotrophic strains with 50 mM CaCl2 at high pH. The hybrids have a morphology different from that of normal haploid strains, but similar to that of aposporously produced diploids. The progeny resulting from selffertilisation of the hybrids show a segregation which is consistent with their being the products of meioses in an autotetraploid.
TL;DR: The tif-1 mutation leads to a temperature sensitive form of protein X which can spontaneously remove the repressor without the intervention of the inducer, suggesting thattif-1 is a mis-sense mutation in the gene coding for protein X.
Abstract: Protein X, molecular weight 40,000, has been separated from the other proteins of E. coliby a two-dimensional gel electrophoretic technique which separates proteins according to isoelectric point (pI) in the firstdimension and according to molecular weight in the second. When protein X is induced in wild-type cells by mitomycin C treatmentit has a pI∼6.0. However, when protein X is induced in a tif-1 mutant, either by temperatureshift-up to 42° or by mitomycin C treatment at 30°, it has a pI∼6.2. The low level of protein X which is present inuninduced tif mutants at 30° also has a pI∼6.2. These results suggest thattif-1 is a mis-sense mutation in the gene coding for protein X. Since transduction andcomplementation studies indicate that tif-1 is a mutation of therecA + gene (Castellazzi, Morand, George and Buttin, 1977) it follows that protein X is the recA+ gene product.
TL;DR: In ccr1 mutants, derepression of isocitrate lyase, fructose-1,6-diphosphatase, ADHII and possibly of the cytoplasmic MDH is prevented, whereas the mitochondrial TCA-cycle enzymes, succinate dehydrogenase and malate dehydrogenases, are not significantly affected.
Abstract: Yeast mutants deficient in the constitutive ADHI (adc1) were used for the isolation of mutants with deficiencies of the intermediary carbon metabolism, and of mutants defective in carbon catabolite derepression. Mutants were recognized by their inability to grow on YEP-glycerol and/or on ethanol synthetic complete medium. They were either defective in isocitrate lyase (icl1), succinate dehydrogenase (sdh1), or malate dehydrogenase (mdh1, mdh2), mdh-mutants could not uniformely be appointed to one of the known MDH isozymes. Homozygous mdh and sdh1 diploids are unable to sporulate.
TL;DR: The results indicate that thecreB andcreC genes may have a regulatory role in the control of carbon catabolism.
Abstract: Mutants altered in carbon catabolite regulation have been isolated by selecting for mutants of theareA217 strain capable of using acetamide as the sole nitrogen source in the presence of sucrose. In addition tocreA mutants described previously by Arst and Cove, strains with mutations in two new genes,creB andcreC, have been found. ThecreB andcreC mutants grow poorly on some sole carbon sources and have low levels of some enzymes of carbon catabolism e.g. β-galactosidase and D-quinate dehydrogenase. ThecreB andcreC mutants are hypersitive to fluoroacetate, fluoroacetamide and allyl alcohol in the presence of glucose or sucrose but not glycerol; and the enzymes, acetamidase, and alcohol dehydrogenase, are less sensitive to carbon catabolite repression than the wild-type strain. Extracellular protease and α-glucosidase enzyme activities are elevated increB andcreC mutants, while L-proline and L-glutamate uptake capacities are lower in both the presence and absence of glucose. Interactions betweencreA, B and C mutations have been investigated in double mutants, and the dominance properties ofcreB andcreC mutants determined. The results indicate that thecreB andcreC genes may have a regulatory role in the control of carbon catabolism.
TL;DR: Results indicate that thednaA gene product is required for the replication of pSC101 DNA.
Abstract: Replication of pSC101 was analyzed by using DNA-DNA hybridization and alkaline sucrose gradient centrifugation. Mutants of thednaA gene were tested for their capacity to replicate pSC101 DNA at a non-permissive temperature. Only a small amount of radioactive precursor was incorporated into pSC101 DNA indnaA mutants at 42°C whereas active incorporation into plasmid DNA took place indnaA
+ strains under the same conditions. The effect of thednaA mutation was grater on plasmid DNA synthesis than on host chromosomal DNA synthesis. The numbers of copies of pSC101 per chromosome in wild type anddnaA strains, grown at 30°C, were about 8 and 2, respectively. These results indicate that thednaA gene product is required for the replication of pSC101 DNA.
TL;DR: The results provide additional evidence that agrocin 84 sensitivity, exclusion of phage AP1, ability to catabolize the guanidine derivatives octopine and nopaline and tumor inducing ability, are Ti-plasmid determined properties.
Abstract: The P type conjugative plasmid RP4 has been shown to be able to promote the transfer of the Agrobacterium Ti-plasmid. The results provide additional evidence that agrocin 84 sensitivity, exclusion of phage AP1, ability to catabolize the guanidine derivatives octopine and nopaline and tumor inducing ability, are Ti-plasmid determined properties. Furthermore, the results strongly support the notion that at least part of the Ti-plasmid is transferred from the bacterium to the target plant cells, since it was demonstrated that Ti-plasmid linked genes specify the synthesis of octopine or nopaline by crown-gall tumor cells.
TL;DR: It is found that the frequency of transposition of Tn801 from RP1 to R388 exceeded, by at least a factor of one hundred, the frequency at which it was deleted fromRP1.
Abstract: We have examined the incidence of loss of the TnA unit, Tn801, from RP1 under conditions where transposition of Tn801 to another replicon. R388, was readily detected. We found that the frequency of transposition of Tn801 from RP1 to R388 exceeded, by at least a factor of one hundred, the frequency at which it was deleted from RP1. We conclude that, in general, transposition of Tn801 does not generate derivatives of the donor plasmid which specifically lack Tn801. The relevance of these findings to the mechanism of transposition is discussed.
TL;DR: The first successful culture, with sustained divisions, of protoplasts from intact plants of Zea mays is described, using a hanging microdrop array technique which permits the testing of very large numbers of different culture media and hormone variations.
Abstract: The first successful culture, with sustained divisions, of protoplasts from intact plants of Zea mays is described. The method involves the use of a hanging microdrop array technique which permits the testing of very large numbers of different culture media and hormone variations. Several different phytohormone combinations were found to allow sustained divisions in protoplasts isolated from stem tissue of corn plants, suggesting the importance of the source of the protoplasts rather than specific medium conditions. In some cases more than 5% of the protoplasts divided, giving macroscopic calluses within 35 days.
TL;DR: The cleavage map of the plasmid RK2 was determined for the five restriction endonucleases EcoRI, HindIII, Bam H-I, Sal I and Hpa I and indicated that genes required for the maintenance of this plasmids in E. coli are not tightly clustered.
Abstract: The cleavage map of the plasmid RK2 was determined for the five restriction endonucleases EcoRI, HindIII, Bam H-I, Sal I and Hpa I. DNA has been inserted into several of these sites and cloned in Escherichia coli. Efforts to obtain derivatives of RK2 reduced in size by restriction endonuclease digestion of the plasmid were not successful and indicated that genes required for the maintenance of this plasmid in E. coli are not tightly clustered. An RK2 derivative possessing an internal molecular rearrangement was obtained by transformation with restriction endonuclease digests of the plasmid.
TL;DR: Shot formation on inductive medium was restored, indicating that the inducibility factor had not been altered in KR103, and genetic complementation and induced regulatory changes due to genome interaction are discussed as possible explanations for the restoration of morphogenic potential.
Abstract: KR103 is a kanamycin resistant cell line of Nicotiana sylvestris, deficient in inducible shoot redifferentiation. The existence of a genetically unaltered inducibility factor in KR103 was tested by fusing protoplasts of this line with those of Nicotiana knightiana, which can not be induced to form shoots in callus culture. Selection of somatic hybrids was based on screening for kanamycin resistance and green pigmentation, factors originally separated in the parental lines. The hybrid nature of two lines (H1 and H2) was shown by studies on esterase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase isoenzymes. In the somatic hybrids shoot formation on inductive medium was restored, indicating that the inducibility factor had not been altered in KR103. Genetic complementation and induced regulatory changes due to genome interaction are discussed as possible explanations for the restoration of morphogenic potential.
TL;DR: Thermosensitive mutants, auxotrophic for ergosterol synthesis, have been isolated, analyzed genetically and their enzymatic deficiencies investigated and seven unlinked complementation groups were classified.
Abstract: Thermosensitive mutants, auxotrophic for ergosterol synthesis, have been isolated, analyzed genetically and their enzymatic deficiencies investigated. These mutants were classified into seven unlinked complementation groups. These groupes lack the following enzymatic activities: squalene epoxidase (erg 1), 2,3-oxidosqualene-lanosterol cyclase (erg 7), phosphomevalonic kinase (erg 8), mevalonic kinase (erg12) and squalene synthetase (erg 9, erg 10, erg 11).