Temperature-sensitive mutations affecting the replication of F-prime factors in Escherichia coli K 12

Summary More temperature-sensitive mutants affecting the replication of the F-gal⁺ episome of Escherichia coli K12 have been isolated. Eight of the mutations were located on F itself and three were located on the chromosome.The temperature sensitive F-gal⁺'s have been integrated into the chromosome to produce Hfr strains. These Hfr strains have transfer origins similar to Hfr Cavalli, and all show aberrant excision and transfer of elongated segments of the chromosome including the integrated F-gal to generate long merodiploids.The chromosomal mutations that govern the replication of F have been termed seg (for segregation). Wild-type F-gal⁺ can be integrated into seg cells at 42° C to give Hfrs, in a process analogous to integrative suppression in the formation of Hfrs from cells carrying mutations that are temperature-sensitive for chromosomal DNA replication (dnaA). A curious feature of an Hfr derived from a seg strain is that it also shows F-genote enlargement as well as normal transfer of chromosomal genetic marker. Preliminary transductional mapping data show that the mutation seg-2 is linked to the threonine locus (minute 0).     

Isolation and mapping of Escherichia coli K12 mutants defective in Tn9 transposition

Summary Five mutants (called tnm) of Escherichia coli with impaired ability for transposition of Tn9 were isolated after treatment with ethyl methanesulfonate (EMS) or N-methyl-N-nitro-N-nitrosoguanidine (NG).The map locations of the tnm mutations were deterimined by a combination of Hfr matings, F episome complementation and P1 transductional mapping. The data obtained show that the five tnm mutations are located near 91 min on the Escherichia coli linkage map and are cotransducible with the metA marker with a frequency of 3%–4%. Introduction of F plasmids containing this region complements the Tnm^- phenotype for the two mutants tested i.e. tnm-1 and tnm-2 are recessive in tnm ⁺/tnm^-merodiploids.     

Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168

Summary We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB: : cat, leuB ^–, and pro: : neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB ⁺ pro⁺)(leuB: : cat pro ⁺) (leuB ^– pro: : neo)(leuB ^– pro ⁺) resulted in a leuB ^– single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD ^– and pro ^–, as well as the double mutation leuB ^– pro ^– can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.     

Mutants in yeast affecting ethidium bromide induced rho- formation and their effects on transmission and recombination of mitochondrial genes.

Summary A series of mutants called ebi, less inducible by ethidium bromide than the parental strain for the ^+– mutation have been isolated after E.M.S. mutagenesis. Some of the ebi mutants also show an important accumulation of ^– cells, in the absence of ethidium bromide. Ebi mutations are nuclearly inherited as shown by meiotic segregation. The effects of these mutants on the transmission and recombination of mitochondrial genes among the diploid progeny of crosses have been studied. Some of the ebi mutants show a non coordinated transmission of the oli1 mitochondrial marker with respect to other mitochondrial markers unexpected for homosexual crosses. This bias which is independent from will be discussed in relation to the segregation and recombination. No significant decrease of the frequency of recombinants has been detected.Abbreviations E.B. Ethidium bromide - E.M.S. Ethyl méthane sulfonate - C^S/C^R Allelic forms of the rib 1 locus conferring chloramphenicol sensitivity/resistance - E^S/E^R Allelic forms of the rib 3 locus conferring erythromycine sensitivity/resistance - O^R/O^R Allelic forms of the oli 1 locus conferring oligomycin sensitivity/resistance - P^S/P^R Allelic forms of the par 1 locus conferring paromomycine sensitivity/resistance - D^S/D^R Allelic forms of the diu 1 or diu 2 loci conferring diuron sensitivity/resistance - C^S/C^R Allelic forms of the mitochondrial locus - ⁺ grande or respiratory competent cells - ^– petite or cytoplasmic respiratory deficient cells     

Detection of antimycin-binding subunits of Complex III by photoaffinity-labeling with an azido derivative of antimycin

Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K _i =ca. 0.5 µM) was considerably less than that of antimycin (K _i 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [³H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [³H]DAA, respectively. The labeling of band 7 by [³H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [³H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding.     

Analysis of ultraviolet light-induced suppressor mutations in the strain of Escherichia coli K-12 AB1157: an implication for molecular mechanisms of UV mutagenesis

Summary Genetic analysis of histidine independent (His⁴) revertants induced by ultraviolet light in the his-4 E. coli strain AB1157 was carried out: 83% carried ochre (UAA) suppressor mutations and 17% carried back mutations to his ⁺ or (intragenic?) suppressors not detectably separable from his-4. Using the specialized transducing psu 2int ^– phage, which carries supE-supB, it was determined that 87% of the ochre suppressors mapped in the supE-supB region. We were able to deduce that 56% of these affected tRNA ₁ ^Gln by a CAATAA change in the tRNA gene while 31% affected tRNA ₂ ^Gln by TAGTAA change. Although we were unable to deduce the base substitution of the remaining 13%, the results indicated that most of the suppressor mutations are caused by a G:C to A:T transition.These results suggest that the high incidence of supE-supB region suppressor mutation in E. coli by UV would be a reflection of the general feature of UV mutagenesis; i.e. preferential induction of G:C to A:T transition in repairing nonparing DNA lesions.     

Unexpected segregation ratios from male-linked translocations in the Mediterranean fruitfly,Ceratitis capitata (Diptera: Tephritidae)

Three male-linked translocations were isolated in the Mediterranean fruitfly, Ceratitis capitata (Wiedemann) which showed sex linkage for an autosomal pupal colour marker, white pupa (wp). The wild type allele (wp ^–) was translocated to the male-determining chromosome. Males emerge from wild type pupae (brown) and females from white pupac. These lines will be used to develop an automatic sexing system.During maintenance of these lines in large populations it was noted that in two of them there was a significant distortion in the expected 1: 1 segregation ratio for pupal colour and excess white pupae were produced. However, adults did not emerge from a large proportion of these pupae. Using known numbers of 1st instar larvae from the three lines, the distortion was further studied and the effect confirmed. To account for this pupal colour segregation distortion it was assumed that certain duplication/deficiency zygotes survive to the late pupal stage. These zygotes have a deficiency for the wild type allele, therefore the normally recessive mutant allele wp could be expressed (pseudo-dominance).The overall fitness of the three lines was calculated, and their suitability for the genetic sexing of large populations is discussed.     

Influencing the spontaneous and chemically induced mutability of E. coli through changing the genetic background

Summary The influence of a second auxotrophic marker to the spontaneous and chemical-induced mutability to prototrophy of a first auxotrophic marker in 7 monoauxotrophs and 31 biauxotrophs of E. coli K 12 was studied by growth layer technics. No case of influence of a second auxotrophy to the mutagen-induced mutability (7 mutagens tested) of the first auxotrophy among 172 possibilities was found. An influence to the spontaneous mutability seemed to be present in 4 cases out of 31. But 3 of them were shown to be imitated by influences of components of the medium to the growth of mutants or parent type. In one case a mutator mutation is responsible for the about 8 times higher rate of the mutation met 1+ in strain met 1/his 7 than in strain met 1. But backmutation and crossing experiments showed that the mutator (mum ⁺) was separate from the auxotrophic marker his 7 (recombination frequency 5/16). This mutator did not increase remarkably the spontaneous mutability of other markers tested (resistence to phages T1 or T4 or to Streptomycin 3, 5, 10, or 100 /ml, or to Chloramphenicol 2 /ml). It is assumed that the spontaneous mutations in the wild-type are at least partially different in their nature from the mutator promoted ones.     

Reduction of cytochromes with menaquinol and sulfide in membranes from green sulfur bacteria

Reduction of cytochromes in chlorosome-free membranes of Chlorobia was studied anaerobically, with an LED array spectrophotometer. For Chlorobium tepidum these membranes contained 0.2 moles cytochrome per mole of bacteriochlorophyll a. The observed change upon complete reduction of oxidized membranes with dithionite could be satisfactorily fitted with three cytochrome components having absorption peaks at 553 (cyt c), 558 and 563 nm (cyt b), in relative amounts of 5:1:2. About 20% of total cytochrome 553 were reducible by ascorbate. Menaquinol reduced all of the 553-component, and this reduction was sensitive to stigmatellin, NQNO and antimycin A. The reduction was insensitive to KCN. However, it was transient at low concentrations of menaquinol in the absence of KCN, but permanent in its presence, demonstrating that electron transport into an oxidation pool was blocked. The 563-component was only slightly reduced by menaquinol unless NQNO or antimycin were present. The stimulation of cytochrome 563-reduction by these inhibitors was more pronounced in the presence of ferricyanide. This phenomenon reflects oxidant-induced reduction of cytochrome b and demonstrates that a Q-cycle is operative in Chlorobia. Also, sulfide fully reduced cytochrome 553, but more slowly than menaquinol. KCN inhibited in this case, as did stigmatellin, NQNO and antimycin A. NQNO was a better inhibitor than antimycin A. Cytochrome 563 again was hardly reduced unless antimycin A was added. The effect was more difficult to observe with NQNO. This supports the conclusion that sulfide oxidation proceeds via the quinone pool and the cytochrome bc-complex in green sulfur bacteria.Abbreviations BChl bacteriochlorophyll - cyt cytochrome - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - SQR sulfide-quinone reductase Dedicated to Prof. Dr. Aloys Wild on occasion of his 65th birthday.     

Specific binding of the calcium channel blocker [3H]verapamil to membrane fractions of Chlamydomonas reinhardtii

Specific binding of the calcium antagonist [³H]verapamil to a microsomal fraction, a presumptive plasma membrane fraction and an intracellular membrane fraction of the phototactic unicellular green alga Chlamydomonas reinhardtii has been demonstrated. The specific activity of the plasma membrane marker enzyme K⁺-stimulated, Mg²⁺-dependent ATPase was severalfold higher in the upper (polyethylene glycol-rich) than in the lower (dextran-rich) phase, and the reverse was established for the marker enzymes of intracellular membranes such as cytochrome c oxidase for mitochondria and antimycin Aresistant NADPH-cytochrome c reductase for endoplasmic reticulum. Chlorophyll as a marker for thylakoid fragments was exclusively found in the lower phase. In the microsomal fraction two specific binding sites of [³H]verapamil were found at 22°C, one with higher and a second with lower affinity to [³H]verapamil. Separation of plasma membranes from intracellular membranes revealed that the highaffinity binding site is attributed to the plasma membrane fraction whereas the low-affinity binding site can be attributed to the intracellular membrane fraction. Specific binding to both separated membrane fractions is saturable and reversible. [³H]Verapamil binding to plasma membranes was not inhibited by the calcium channel blockers diltiazem and nifedipine. However, in the intracellular membrane fraction [³H]verapamil could be displaced by diltiazem but not by nifedipine. Increasing concentrations of calcium chloride inhibited [³H]verapamil binding in both fractions.Abbreviations B_max maximum density of binding sites - BSA bovine serum albumin - Cyt.c cytochrome c - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-amino-ethylether)N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IC₅₀ concentration causing 50% inhibition - Mes [N-morpholino]ethanesulfonic acid - PEG polyethylene glycol - PMSF phenylmethylsulfonylfluoride - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid     

Polarity of localised conversion in Streptococcus pneumoniae transformation

Summary Localised conversion in pneumococcal transformation is a process that spans a few nucleotides when the 5-ATTAAT/3-TAAGTA configuration occurs at the pairing step. It was first observed in two-point crosses between an amiA mutation (amiA36) carrying this sequence and other closely linked mutants of the locus. The yield of the amiA resistance allele conversion to wild type is 20%. In order to characterize this process, which differs from longpatch conversion by the length of DNA repair, gene requirements and sequence specificity, we devised expreriments to detect the reciprocal conversion, AmiA⁺ to AmiA^r. For this purpose we examined the suppressibility by a pneumococcal informational suppressor of several nonsense mutations at the locus. Amber (UAG) and ochre (UAA) mutations are suppressed whereas UGA is not suppressed. In this genetic background, where amiA36 is partly suppressed, it was possible to select for double mutants in a cross between amiA36 and a closely linked non-suppressible marker. Direct isolation of such double mutants was also performed without any screening in crosses between amiA36 and the same linked marker in cloned DNA. The frequency of double mutants was very low (1/175) suggesting that there is no conversion of wild-type to mutant alleles. Thus conversion is a polarized process changing specifically A to C.     

Genetic analysis and molecular cloning of the Escherichia coli ruv gene

Summary The genetic organisation of the ruv gene, a component of the SOS system for DNA repair and recombination in Escherichia coli, was investigated. New point mutations as well as insertions and deletions were generated using transposon Tn10 inserted in eda as a linked marker for site specific mutagenesis, or directly as a mutagen. The mutations were ordered with respect to one another and previously isolated ruv alleles by means of transductional crosses. The direction of chromosome mobilization from ruv:: Mud(Ap^R lac)strains carrying F42lac ⁺ established that ruv is transcribed in a counterclockwise direction. Recombinant phages able to restore UV resistance to ruv mutants were identified, and the ruv ⁺ region was subcloned into a low copy number plasmid. The ruv ⁺ plasmid was able to correct the extreme radiation sensitivity and recombination deficiency of ruv recBC sbcB strains.     

Genetic mapping by means of protoplast fusion in Bacillus subtilis

Summary A new mapping method involving protoplast fusion in Bacillus subtilis is described. Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, x, to be mapped were fused with polyethylene glycol, and purA ⁺ purB ⁺ fusants were selected. After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers. This method was fully applicable to PBS1-resistant strains (e.g., lyt strains). The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152). The properties of lyt-15 are also discussed.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - SMM 0.5 M sucrose, 0.02 M MgCl₂, 0.02 M maleate buffer, pH 6.5     

The Kavar(D) dominant female-sterile mutations of Drosophila reveal a role for the maternally provided alpha-tubulin4 isoform in cleavage spindle maintenance and elongation

The dominant-negative female-sterile Kavar^D mutations and their revertant kavar^r alleles identify the Tubulin67C gene of Drosophila melanogaster, which codes for the maternally provided -tubulin⁴ isoform. The mutations result in the formation of monopolar, collapsed spindles (each with two nearby centrosomes, a tassel of microtubules and overcondensed chromosomes), thus revealing a novel function for -tubulin⁴ in spindle maintenance and elongation. Molecular features of the two Kavar^D alleles and a kavar^null allele are described and models for their actions are discussed.     

Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after “gamma”-fusion

Summary Extremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt⁶⁰,100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via gamma-fusion.     

Mobilization of the transposable element mdg4 by hybrid dysgenesis generates a family of unstable cut mutations in Drosophila melanogaster

Summary A family of unstable mutations at the cut locus in Drosophila melanogaster was obtained under the conditions of hybrid dysgenesis (Gerasimova 1981, 1982). The in situ hybridization experiments have shown that, in the original unstable ct ^MR2 mutation, the 7B region of the X chromosome (where cut is located) contains a mobile dispersed genetic element, mdg4. All other unstable ct mutations derived from ct ^MR2 including visible and lethal alleles and unstable ct ⁺ reversions, also contain mdg4 in the 7B region. The X chromosomes of the parent strain (wild type) do not contain mdg4 at all. All stable revertants derived from ct ^MR2, from other unstable ct mutations, or from ct lethals lost mdg4 from the 7B region. The ct ^MR2 X chromosome does not contain P-elements, although a few copies are present in the autosomes. The instability of the ct ^MR2./ct ^MR2 strain remained at a high level for 50 generations (1.5 years) and then rapidly decreased. A new cross with an MRh12/Cy strain (originally used for dysgenesis induction and containing a number of P-elements) increased the instability to a level exceeding the original one. The data strongly suggest that unstable ct mutations in our system are induced by transpositions of mdg4, possibly activated by P-elements.     

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster

Summary The vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr ⁺) and excision-repair-deficient (exr ^–) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr ⁺ strain and 24 from the exr ^– strain, were characterized by sequence analysis. In two mutants obtained from the exr ⁺ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr ⁺ strain, 22 (76%) were GCAT transitions, 3 (10%) ATTA transversions, 2 (6%) GCTA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GCAT transitions. Of the mutations in an exr ^– background, 12 (48%) were GCAT transitions, 7 (28%) ATTA transversions, 5 (20%) GCTA transversions and 1 (4%) was a ATGC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.     

Long range control circuits within mitochondria and between nucleus and mitochondria

Summary To uncover the functional circuitry both within the mitochondrial genome and between the mitochondrial and the nuclear genome, we have developed a general method for selecting and characterizing genetically suppressor mutations that restore the respiratory capacity of mit ^- mitochondrial mutants.Several hundreds of pseudo-wild type revertants due to a second unlinked mutation which suppresses a target mit ^- mutation were isolated. The suppressor mutations were found located either in the nuclear (abbreviated NAM for nuclear accommodation of mitochondria) or in the mitochondrial genome (abbreviated MIM for mitochondrial-mitochondrial interaction).The specificity of action of various suppressors upon some 250 different mit ^- mutations located in several genes was tested. According to this specificity of action, suppressors were subdivided into two major classes: allele specific or gene specific suppressors. Because the cob-box mitochondrial gene has a mosaic organization, we were able to find a novel third class of extragenic suppressors specific for mit ^- mutations within the introns of this gene.Four examples of suppressors showing various specificities of action illustrate our approach. (1) a nuclear gene controlling specific alleles of different mitochondrial genes; (2) a nuclear gene controlling selectively one intron of a split mitochondrial gene; (3) a mitochondrial gene controlling specific alleles of different mitochondrial genes; (4) a region in one complex mitochondrial gene which controls selectively one intron of another split mitochondrial gene.Different mechanisms of suppression are discussed stressing the alleviation of splicing deficiencies of intron mutations.     

Molecular cloning of the T4 genome: organization and expression of the tail fiber gene cluster 34--38

Summary T4 derivatives that carry T4 tail fiber genes 34–38 have been isolated and characterized by genetic, structural and functional analysis. 32 T4 recombinants were identified by a marker rescue screen of 310 T4 clones generated by restriction of partial cytosine-containing T4 DNA with either HindIII or EcoRI and ligation into appropriately cleaved vectors. These tests defined 15 recombinant classes with respect to the contiguous stretches of genome recovered. Restriction enzyme structural analysis identified 7 HindIII fragments and 7 EcoRI fragments, established a restriction map covering about 11 kb, and indicated the orientation of the DNA inserts within the vectors. The cloned tail fiber genes are expressed efficiently from promoters and complement in vivo T4 phage carrying amber mutations in the tail fiber genes. Polypeptides corresponding to gp34-gp38 have been detected by SDS polyacrylamide gel electrophoresis of ³⁵S-labeled extracts of appropriate T4 recombinant infected UV-treated host cells. The genetic, structural and functional maps of the T4 tail fiber gene cluster have been correlated, and provide a rational approach to genetically directed DNA sequence analysis of genes 34–38 and their mutant variants that affect the assembly, structure and function of the tail fibers.     

Segregation and re-segregation of an extranuclear gene in Chlamydomonas reinhardii

Summary Streptomycin-sensitive revertants (=prim. ss) result from the streptomycin-dependent mutant sd ₃ with a frequency of 10^-7 to 10^-6 by mutation. The newly formed ss-revertants have besides ss-genes also sd-genes. Therefore, most of the revertants are able to segregate sd-cells (=sec. sd) on streptomycin-medium up to a frequency of 10^-2. With increasing time of cultivation on streptomycin-free medium the number of the sd-genes decreases continuously until the power of segregation expires.The newly formed sec. sd-cells possess besides the sd-genes also ss-genes. So they are also able to segregate ss-revertants with a frequency of 10^-2. However, the power of segregation of the sec. sd-mutants decreases much faster than the power of segregation of the primarily formed ss-revertants. Thus the ss-genes are blocked much more by streptomycin than the sd-genes by antibiotic-free medium. Very soon the sec. sd-cells possess only sd-gene copies and become identical with the mutant sd ₃.The origin of sec. ss-cells from sec. sd-clones is caused not only by segregation but also by mutation sdss which is also responsible for the development of primarily ss-cells from the sd-mutant. Therefore, the origin of sec. ss-cells from sec. sd-clones never reach the absolute zero-level. On the other hand the mutations sssd, resp., ss-revertant sd never happen spontaneously.New sec. ss-revertants possess the ability to form tertiary sd-cells.It is suggested, that the gene sd ₃ is more likely to be localized in the mitochondria than in the chloroplast.

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Vorgelegt von G. Melchers