Showing papers on "Mutant published in 1979"

An adenovirus type 5 early gene function regulates expression of other early viral genes

Abstract: We have identified an adenovirus type 5(Ad5) early gene function located in early region 1 which is required for the production of early cytoplasmic mRNAs corresponding to early regions 2, 3, and 4. Mutant dl312 (lacks the segment between 1.5 and 4.5 map units) grows as well as wild-type virus in 293 cells (Ad5-transformed human embryonic kidney cells), but its growth is severely restricted in HeLa cells. We detect no viral RNAs in the cytoplasm of dl312-infected HeLa cells. Viral RNA sequences are present, however, in dl312-infected HeLa cell nuclei.

In vitro packaging of lambda and cosmid DNA.

Abstract: Publisher Summary This chapter discusses the in vitro packaging of λ and cosmid deoxyribonucleic acid (DNA). The efficiency of cloning a desired DNA fragment in bacteriophage λ depends on a number of factors such as the availability of a useful λ vector, the probability of obtaining a DNA fragment in a possibly enriched form, the efficiency of conversion of the ligated hybrid DNA molecules to plaque-forming particles, and the availability of a suitable probe for the detection of the hybrid phage. The λ and cosmid DNA, as well as in vitro recombinants, are conveniently packaged using a combination of two lysates, each of which is defective in another step of λ morphogenesis. Empty precursor particles accumulate after induction in bacteria containing a prophage mutant in gene D . This mutation can be complemented by the addition of the missing D protein, most effectively by the supply of an induced λ E – lysogen. This mutation affects the main capsid protein; in its absence, all other head proteins are available in a soluble form. In the presence of adenosine triphosphate (ATP), spermidine, and putrescine, DNA is packaged, the head matures, and tails—components of both lysates—are attached. The result is a DNase-resistant infectious particle, which can be stored like any in vivo -produced phage.

The beige mutation in the mouse selectively impairs natural killer cell function.

Abstract: GERM LINE mutations affecting defined cell populations are often valuable tools in elucidating the function of these cells in complex biological systems such as tumour rejection. We report here that a mutant gene in the mouse called beige (bgJ), leads to a complete and selective impairment of naturally occurring killer lymphocytes, whereas all other forms of cell-mediated lysis are apparently normal. The defective gene product may lie within the lytic pathway subsequent to tumour cell contact. Because many cell types, including natural killer (NK) cells, T cells and macrophages, may be involved in tumour resistance in vivo1, these mice will provide a critical test of the hypothesis that it is NK cells which provide a first line of defence against neoplasia2. It is likely that this mutant will be invaluable for further investigations in tumour immunology just as the nude mouse has been indispensable in evaluating the role of thethymus in the development of the T-lymphoid system and the role of T cells in the rejection of tumours.

Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): Two gene systems control movement

Abstract: A large number of motility mutants of the gliding bacterium Myxococcus xanthus have been isolated and analyzed by transduction Almost all nonmotile mutants are found to be double mutants This is explained by the existence of two parallel and almost independent multi-gene systems controlling motility, in which case at least one mutation in each system would be required eliminate motility Only one locus, called mgl, has been found to be shared by both systems Wild type cells move singly and in groups Single cells move if they carry a complete gene system A, the genes of which are described in the preceding paper Groups of cells can move if they carry a complete gene system S, but single A−S+ cells do not move Linkage analysis reveals at least 9 different loci in system S One class of S− mutants, those mutated in the locus tgl, are conditional mutants which, after contact with tgl + cells, become temporarily motile as cell groups Most system A mutations have little effect on fruiting but many system S mutations block it, suggesting that system S plays a role in the fruiting process

Escherichia coli Mutants Thermosensitive for Deoxyribonucleic Acid Gyrase Subunit A: Effects on Deoxyribonucleic Acid Replication, Transcription, and Bacteriophage Growth

Abstract: Temperature-sensitive nalA mutants of Escherichia coli have been used to investigate the structure and functions of deoxyribonucleic acid (DNA) gyrase. Extracts of one such mutant (nalA43) had thermosensitive DNA gyrase subunit A activity but normal gyrase subunit B activity, proving definitively that nalA is the structural gene for subunit A. Extracts of a second nalA (Ts) mutant (nalA45) had a 50-fold deficiency of gyrase subunit A activity. The residual DNA supertwisting was catalyzed by the mutant DNA gyrase rather than by a novel supertwisting enzyme. The nalA45(Ts) extract was also deficient in the nalidixic acid target, which is defined as the protein necessary to confer drug sensitivity to in vitro DNA replication directed by a nalidixic acid-resistant mutant extract. Thus, gyrase subunit A and the nalidixic acid target are one and the same protein, the nalA gene product. Shift of the nalA43(Ts) mutant to a nonpermissive temperature resulted in a precipitous decline in the rate of [(3)H]thymidine incorporation, demonstrating an obligatory role of the nalA gene product in DNA replication. The rates of incorporation of [(3)H]uridine pulses and continuously administered [(3)H]uracil were quickly reduced approximately twofold upon temperature shift of the nalA43(Ts) mutant, and therefore some but not all transcription requires the nalA gene product. The thermosensitive growth of bacteriophages phiX174 and T4 in the nalA43(Ts) host shows that these phages depend on the host nalA gene product. In contrast, the growth of phage T7 was strongly inhibited by nalidixic acid but essentially unaffected by the nalA43(Ts) mutation. The inhibition of T7 growth by nalidixic acid was, however, eliminated by temperature inactivation of the nal43 gene product. Therefore, nalidixic acid may block T7 growth by a corruption rather than a simple elimination of the nalidixic acid target. Possible mechanisms for such a corruption are considered, and their relevance to the puzzling dominance of drug sensitivity is discussed.

Guanine nucleotide-binding activity as an assay for src protein of rat-derived murine sarcoma viruses

Abstract: We have recently identified a 21,000-dalton protein, p21, coded for by Kirsten or Harvey murine sarcoma virus. On the basis of the results obtained with the p21 of a mutant of Kirsten sarcoma virus, temperature sensitive for the maintenance of transformation, we concluded that the p21 was required for the maintenance of transformation induced by either virus. We report herein that when extracts from cells transformed by Kirsten or Harvey sarcoma virus are incubated with [3H]GDP or [α-32P]GTP, picomole quantities of guanine nucleotide can be immunoprecipitated with antisera that contain antibodies to the p21. Previously we have shown that the immunoprecipitability of [35S]methionine-labeled p21 of the temperature-sensitive mutant of Kirsten sarcoma virus is thermolabile. The binding of guanine nucleotide is shown herein also to be thermolabile in extracts of cells transformed by the same mutant. However, the immunoprecipitability of the [35S]methionine-labeled p21 in such extracts of the temperature-sensitive mutant can be preserved if the extracts containing labeled p21 are incubated with added GDP or GTP prior to heating. The results suggest an interaction between p21 and certain guanine nucleotides, and the possible roles of guanine nucleotides and p21 in the maintenance of transformation are discussed.

Albumin-deficient rat mutant

Abstract: An analbuminemic colony was established from Sprague-Dawley rats. Analbuminemia was inherited as an autosomal recessive trait. The rates of growth and reproduction of the mutant rats were no different from those of normal rats. Biochemically, the mutant was characterized by an extraordinarily low serum albumin content and a hyperlipidemia. Total serum protein in the mutant rat was similar to that of control Sprague-Dawley rats, with increased globulin. Serum cholesterol was inversely correlated with a decrease in albumin; the correlation coefficient for ablumin was --.92. These mutant rats may serve as a model of human familial analbuminemia and may also be useful in elucidating the functional roles of albumin.

Identification of a Gene for a-Tubulin in Aspergillus nidulans

Abstract: N. Ronald Morris, Margaret H. Lai and C. Elizabeth Oakley Department of Pharmacology CMDNJ-Rutgers Medical School Piscataway, New Jersey 08854 Summary This paper demonstrates that revertants of tem- perature-sensitive benA (p-tubulin) mutations in Aspergillus nidulans can be used to identify pro- teins which interact with /3-tubulin. Three beno- myl-resistant benA (P-tubulin) mutants of Asper- gillus nidulans, BEN 9, BEN 15 and BEN 19, were found to be temperature-sensitive (ts-) for growth. Temperature sensitivity co-segregated with benomyl resistance among the progeny of outcrosses of BEN 9, 15 and 19 to a wild-type strain, FGSC#99, indicating that temperature sensitivity was caused by mutations in the benA gene in these strains. Eighteen revertants to ts+ were isolated by selection at the restrictive tem- perature. Four had back-mutations in the benA gene and fourteen carried extragenic suppressor mutations. Two of the back-mutated strains had p-tubulins which differed from the p-tubulins of their parental strains by one (l-) or two (2-) negative charges on two-dimensional gel electro- phoresis. Although the P-tubulins of the extra- genie suppressor strains were all electrophoreti- tally identical to those of the parental strains, one of the suppressor strains, BEN 9R7, had an electrophoretic abnormality in al-tubulin (1). A heterozygous diploid between this strain and a strain with wild-type al-tubulin was found to have both wild-type and mutant (l+) rul-tubulins. This experiment rules out post-translational modifica- tion as a possible cause of the al-tubulin abnor- mality. Thus the suppressor mutation in BEN 9R7 must be in a structural gene for al-tubulin. We propose that this gene be designated tubA to denote that it is a gene for cul-tubulin in A. nidulans. Introduction The assembly of tubulin into microtubules involves it in a large number of a cellular processes. Al- though a great deal is known about microtubules both morphologically and biochemically, there are large gaps in our knowledge. For example, micro- tubule assembly and dynein-dependent microtu- bule-associated movement have been extensively studied in vitro, but almost nothing is known about the regulation of microtubule assembly, disassem- bly or microtubule-dependent transport in the liv- ing cell (Snyder and McIntosh, 1976); and although the molecular architecture of microtubules has been the subject of elegant ultrastructural investi- gation (Snyder and McIntosh, 1976), the molecular details of the interactions between LY- and P-tubu- lins and microtubule-associated proteins are un- known. The genetics of tubulin and microtubules have also not been explored. This paper demon- strates that genetic studies can be used to provide information about the interactions between micro- tubule proteins. Mutants of the fungus Aspergillus nidulans se- lected for resistance to the antimitotic benzimida- zole compounds benomyl or thiabendazole may carry a mutation at any one of three loci, benA, B or C (Van Tuyl, 1977). In a previous paper, we showed that most benA mutants have electropho- retically abnormal p-tubulins and that benA is a structural gene for P-tubulin in A. nidulans (Sheir- Neiss, Lai and Morris, 1978). The present paper extends the potential usefulness of the benA sys- tem for analyzing tubulin structure and function by isolating and characterizing a number of revertants and suppressors of benA mutations. Since the original benA mutants were drug-resistant, it was not possible to isolate revertants directly, but be- cause most of the mutants studied electrophoreti- tally appeared to have missense mutations in the benA gene and because missense mutations may result in destabilization of protein structure, we tested a series of benA mutants for their ability to grow at elevated temperatures with the expectation of finding one or more temperature-sensitive (ts-) strains. Three such ts- mutants were found and characterized, from these a series of revertants to ts+ have been isolated. Among 18 revertants from ts- to ts+, four have intragenic back-muta- tions in p-tubulin and 14 have extragenic suppres- sor mutations. One of the extragenic suppressor strains has electrophoretically abnormal al-tubu- lit-r. The suppressor mutation this strain has been identified as a structural gene for cul-tubulin in A. nidulans. Results BEN 9,155 and 19 Are Temperature-Sensitive Twenty six benA mutants of A. nidulans originally isolated by Van Tuyl (1977) were tested for inability to grow at high temperature. The three strains BEN 9, 15 and 19, carrying the mutations benAll, benA and benA21, were found to be tempera- ture-sensitive (ts-) for growth at 44°C. At this tem- perature, the wild-type parental strain B3 and other benA mutants grew about half as rapidly at 32”C, whereas the three ts- strains were inhibited by -90% (Figure 1). To determine whether the tem-

Genetics of Gliding Motility in Myxococcus xanthus (Myxobacterales): Genes Controlling Movement of Single Cells

Abstract: M. xanthus is a gliding bacterium whose motility is subject to intercellular control. Strain DK101 of M. xanthus gives rise to 6 distinct types of nonmotile mutants and transduction of motility between mutants, mediated by the generalized transducing phage Mx8, identifies the gene loci that underlie the six types. Five of the types, B, C, D, E, and F, are contitional mutants that can be stimulated to move by wild-type cells or by cells of a different mutant type. Mutants of each stimulation type lie in separate and distinct loci, cglB, cglC, cglD, cglE and cglF. The sixth mutant type can stimulate any of the five other types to move, never moves itself, and is produced by mutations in at least 17 loci.

Phenotypic suppression of nonsense mutants in yeast by aminoglycoside antibiotics

Abstract: STREPTOMYCIN, an aminoglycoside antibiotic, can reverse the mutant phenotypes of many nonsense and missense mutations in Escherichia coli and in bacteriophage T4. This phenomenon has been called phenotypic suppression, since the mutant phenotype returns after removal of the drug1. The most likely explanation for phenotypic suppression is that streptomycin promotes mistranslation in vivo, and that acceptable amino acids are inserted into the growing polypeptide chain at the site of the mutant codon. Consistent with this view is the observation that streptomycin causes E. coli ribosomes to mistranslate RNA in vitro2,3. Streptomycin and neomycin have however been found to have no effect in stimulating ribosomes from eukaryotic cells to mistranslate RNA in vitro4,5. A subclass of the aminoglycoside antibiotics has been shown6,7 to stimulate eukaryotic ribosomes to misread RNA. The highly active molecules are distinguished in that they contain the drug fragment paromamine (or 3′-deoxyparomamine). We have therefore examined the capacity of various aminoglycosides to suppress mutations phenotypically in the eukaryotic yeast, Saccharomyces cerevisiae. The results presented here show that paromomycin, which contains paromamine, is capable of phenotypic suppression of the nonsense mutations in S. cerevisiae.

Epistatic gene interactions in the control of division in fission yeast.

Abstract: THERE is currently much interest in the mechanism which controls the timing of cell division. Certain features of the control have been found to be common to a variety of eukaryotes. In particular, the importance of cell size as a parameter affecting cell cycle progress has been reported for mammalian cells(1,2) and for several single-celled eukaryotes(3-6). Another feature common to several systems is that growth conditions have a direct effect on the timing of division cycle events(7-9), and on cell size(9,10). In the fission yeast Schizosaccharomyces pombe, both cell size(6) and nutritional conditions(9) have been shown to affect cycle kinetics. The organism has been used extensively as a model eukaryotic system, largely because of the ease of measuring cell size and because division occurs by binary fission(11). More recently, its genetic tractability has led to the isolation of cell division cycle (cdc) mutants(12), and also of wee mutants altered in the control coordinating growth with the division cycle(13-15). The existence of such control mutants allows a more direct approach to the investigation of the molecular basis of division control, in contrast to the indirect methods used in other systems(4,16-18). wee mutants are so far unique to S. pombe. The most conspicuous property of wee mutants is their reduced cell size(13,14). Analysis of these mutants(15,19) and other evidence(9) has shown that control over cell division timing normally acts at entry to mitosis. As the function of a number of cdc genes is specifically required for mitosis(12), interactions between wee and cdc mutants which affect mitosis might be expected. I report here that the mitotic defect caused by a defective cdc25 allele is suppressed in wee mutants. Suppression by wee1 mutants is almost complete, while the wee2.1 mutation is a less effective suppressor. The significance of these findings for genetic models of the control of mitosis is considered.

Deletions Generated by the Transposon Tn10 in the srl recA Region of the ESCHERICHIA COLI K-12 Chromosome

Abstract: A negative regulatory gene for the srl operon (srlR) was recognized by the characteristics of an insertion mutation generated by the transposon Tn10 determining tetracycline resistance. This finding is discussed in light of previous hypotheses on the regulation of the srl genes, which mediate metabolism of glucitol (i.e., sorbitol). Mapping showed that the order of genes in this region is: srlR srlD srlC recA alaS. Using two different methods, five mutations of both srl and recA were detected. The phenotype conferred by these mutations, UV sensitivity and extreme recombination deficiency, is characteristic of standard recA point mutants. Three of the mutations were deletions that also removed the genes for tetracycline resistance of the nearby transposon. A fourth mutation ended at a distance from Tn10 sufficient to allow separation of the two by recombination following P1 transduction; our tests did not allow us to conclude whether this mutation was an inversion or a deletion. The fifth mutation was a deletion that seemed to end immediately adjacent to the boundary of Tn10, proximal to recA. Mechanisms for the generation of these srl recA mutations are discussed.

Mutant single-strand binding protein of Escherichia coli: genetic and physiological characterization.

Abstract: A mutation in the Escherichia coli gene for single-strand binding protein results in temperature-sensitive deoxyribonucleic acid replication (R. R. Meyer, J. Glassberg, and A. Kornberg, Proc. Natl. Acad. Sci. U.S.A. 76:1702-1705, 1979). The mutant (ssb-1) is also more sensitive to ultraviolet irradiation and about one-fifth as active in recombination. Single-strand binding protein is thus implicated in repair and recombination as well as in replication. The mutation in ssb is located between uvrA and melA at 90.8 min on the genetic map. The ssb gene appears to be allelic with lexC, a gene with a proposed role in regulating inducible deoxyribonucleic acid repair.

Evidence that the avian sarcoma virus transforming gene product is a cyclic AMP-independent protein kinase

Abstract: The avian sarcoma virus transforming gene product pp60src has been partially purified by using ion exchange or immunoaffinity chromatography. These preparations contain a cyclic AMP-independent protein kinase activity capable of transferring radiolabel from [gamma-32P]ATP to immune rabbit IgG, casein, and the heavy chain purified from normal rabbit IgG. The casein kinase activity is specifically inhibited by anti-pp60src IgG. Comparison of the pp60src-protein kinase isolated from cells transformed by a wild-type ASV or by an ASV temperature-sensitive transformation mutant revealed that the latter product had a half-life of 3 min at 41 degrees C whereas that of the wild-type product was 20 min.

A phosphoglycolate phosphatase-deficient mutant of Arabidopsis

Abstract: CURRENT concepts of photosynthetic and photorespiratory carbon metabolism have been derived almost entirely from the results of biochemical and physiological investigations. To examine these concepts by genetic analysis, we have isolated several mutants of a C3 species with defects in CO2 assimilation and photorespiration. We report here the isolation and preliminary characterisation of the first of these, a mutant deficient in phosphoglycolate phosphatase activity.

Five TGA “stop” codons occur within the translated sequence of the yeast mitochondrial gene for cytochrome c oxidase subunit II

Abstract: A mitochondrial mutation that genetically maps in the middle of the gene coding cytochrome c oxidase subunit II has been found to be a single-base-pair deletion. Three independently isolated spontaneous revertants of this mutant have different single-base-pair insertions within 15 nucleotides of the mutation. These findings clearly identify the location of the gene and suggest that the mutation causes a frame-shift. The sequence of about 900 base pairs surrounding the mutation has been determined and found to have several chain termination codons in every possible reading frame. The sequence can, however, be translated in one frame by assuming that the codon TGA does not cause chain termination in yeast mitochondira, as was recently suggested for the human organelle [Barrell, B. G., Bankier, A. T. & Drouin, J. (1979) Nature (London), in press]. If TGA codes for tryptophan residues, as is apparently the case in human mitochondria, a polypeptide can be read from the yeast mtDNA that is identical to bovine cytochrome oxidase subunit II at 37.8% of its residues. Furthermore, the DNA sequences of the frame-shift revertants discussed above predict relative isolectric point differences between the wild-type and various revertant forms of the polypeptide. The detection of these isolectric point differences by two-dimensional electrophoresis of subunit II from the various strains independently confirms the presumed reading frame of the gene. It is concluded that TGA is translated in yeast mitochondria, most probably as tryptophan.

B6.C-H-2bm12. A new H-2 mutation in the I region in the mouse.

Abstract: The B6.C-H-2bm12 mutant is described and evidence is presented for the mutational site occurring in the IA subregion. The mutant is of the gain and loss type as bm12 in equilibrium or formed from C57BL/6 grafts are rejected in 14-16 d. Mapping studies by the gene-complementation method using H-2 recombinant strains place the mutation in the K or IA regions of the H-2 complex and furthermore, the use of this test and the use of other H-2 mutants indicate that H-2Kb is not the site of the mutation, making the IA region the most likely site. Serological analysis with a battery of H-2b, Iab, and other Ia sera, both by cytotoxicity, rosetting, and also by absorption analysis, indicated no alteration in H-2 specificities, particularly in H-2.K33. By contrast, all of the Iab specificities coded for by the IA subregion (Ia.3, 8, 9, 15, and possibly 20) are extensively altered and are either absent or greatly reduced in amount indicating an extensive alteration in the Ia-bearing molecule. The bm12 mutant strongly stimulates the parental C57BL/6 strain in an mixed lymphocyte reaction (MLR), and the reciprocal also occurs, the degree of stimulation being similar to that obtained with K + IA differences originating in another H-2 haplotype and points to the mutation effecting the Lad-1 locus. The presence of an extensive histocompatibility change, a marked alteration in the serologically detected Ia specificities, and a strong MLR, all produced by the one mutation, provides strong evidence for the identity of the Ia-1, Lad-1, and H-2(IA) loci in the IA subregion. The bm12 mutant should be of value in determining the relationship of Ia specificities, Ir genes, and other phenomena effected by the I region.

The CAN1 locus of Saccharomyces cerevisiae: fine-structure analysis and forward mutation rates.

Abstract: A system of strains and growth media was developed to allow efficient detection of forward mutation, reversion, complementation, and suppression at the canavanine-resistance (CAN1) locus of Saccharomyces cerevisiae. Genetic fine-structure analysis revealed that the map length is at least 40, and possibly as much as 60 X-ray map units; this is the longest gene map yet reported in S. cerevisiae. Allelic complementation was not observed, despite testing of a large number of allele pairs, and alleles suppressible by the ochre suppressor SUP11 were absent from a sample of 48 spontaneous mutants and occurred infrequently (7%) among a sample of ultraviolet-induced mutants. Infrequent mutant types included canavanine-resistant mutants capable of arginine uptake and alleles thought to represent deletions or inversions. In contrast to previous reports in the literature, the spontaneous forward mutation rate at CAN1 did not increase during meiosis.

Splicing as a requirement for biogenesis of functional 16S mRNA of simian virus 40.

Abstract: Simian virus 40 deletion mutants were constructed lacking specifically the intervening sequences for a late viral mRNA. The construction method involved the replacement of portions of the late simian virus 40 genes with the DNA segment from reverse transcription of the viral mRNAs. Restriction endonuclease cleavage and sequence analysis confirmed the precise structure of the mutant DNAs and demonstrated that they contained the genetic information for VP1, including all potential 5' ends for the late viral RNAs. Thus, the primary late transcription product(s) of this mutant should have the structure of functional 16S mRNAs. Complementation analysis as well as immunoprecipitation showed, however, that deletion of the intervening sequences from this mutant prevented the expression of VP1. The nature of this failure appears to be a defect in the posttranscriptional processing of the viral RNA. These results indicate that splicing is an essential function in the biogenesis of certain mRNAs.

A response of protein synthesis to temperature shift in the yeast Saccharomyces cerevisiae.

Abstract: When Saccharomyces cerevisiae are subjected to a sudden increase in temperature (22 degrees C to 37 degrees C) they undergo extensive and, in some cases, extreme alterations in their rates of synthesizing individual polypeptides. These changes were monitored by pulse-labeling cells with [35S]methionine and separating the total soluble proteins by two-dimensional gel electrophoresis. Incorporation of 35S into individual proteins was measured by a computer-coupled autoradiogram-scanning method. The rates of synthesis of most proteins are transiently changed; 10-fold or greater induction or respression is common. This temperature response has also been studied in a mutant strain that is temperature sensitive for the nucleus-to-cytoplasm transport of RNA. In this mutant, not only the induction, but also a part of the repression in response to temperature upshift is largely inhibited. Conceivable mechanisms are discussed.

An Escherichia coli mutant defective in single-strand binding protein is defective in DNA replication

Abstract: An Escherichia coli mutant, temperature-sensitive for DNA synthesis in vivo and in vitro, is defective in single-strand binding protein (SSB; DNA-binding protein). Conversion of phage G4 single strands to the duplex form is defective in crude enzyme fractions of the mutant and is complemented by pure wild-type SSB. Radioimmunoassays of mutant extracts show normal levels of material crossreacting with anti-SSB antibody. SSB purified to homogeneity from the mutant is active, with lower specific activity, in the reconstituted G4 replication assay at 30 degrees C, but virtually inactive at 42 degrees C. Surprisingly, the mutant protein, like the wild-type protein, survives heating at 100 degrees C. Thus, mutant SSB is structurally heat-resistant but is functionally thermosensitive in vitro and in vivo. Both the in vivo and in vitro defects are tightly linked in transductions by phage P1. The mutation in the binding protein, designated ssb-1, is located between 90 and 91 min on the E. coli genetic map.

Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures.

Abstract: Mitochondrial (Mt) DNA from mitochondrial mutants of race s Podospora anserina and from senescent cultures of races s and A was examined. In mutants, we observed that fewer full length circles (31 mu) were present; instead, smaller circles characteristic for each mutant studied were found. Eco R1 digestion of these mutant MtDNAs indicated that in certain mutants, although specific fragments were absent, the total molecular weight of the fragments was not much different than wild-type. The properties of senescent MtDNA was strikingly different from either wild-type or mutant Mt DNA. First, a multimeric set of circular DNA was observed for both race s and A, with a monomeric repeat size of 0.89 mu. These circles ranged in size from 0.89 mu to greater than 20 mu; only one molecule out of some 200 molecules was thought to be of full length (31 mu). Density gradient analysis showed that there were two density species: a majority were at the same density as wild-type (1.694 g/cm3) and a second at 1.699 g/cm3. Most of the circular molecules from MtDNA isolated by either total DNA extraction or by extraction of DNA from isolated mitochondria were contained in the heavy DNA fraction. Eco R1 enzymatic digestion indicated that the light DNA had several fragments (amounting to about 23 x 10(6) daltons) missing, compared with young, wild-type MtDNA. Heavy senescent MtDNA was not cleaved by Eco R1. Analysis with Hae III restriction endonuclease showed also that light senescent MtDNA was missing certain fragments. Heavy MtDNA of average size 20 x 10(6) daltons, yielded only one fragment, 2,500 bp long, by digestion with Hae III restriction endonuclease. Digestion of heavy DNA with Alu I enzyme yielded 10 fragments totalling 2,570 bp. By three criteria, electron-microscopy, Eco R1 and Hae digestion, we conclude that the heavy MtDNA isolated from senescent cultures of Podospora anserina consisted of a monomeric tandemly repeating subunit of about 2,600 bp length. These results on the properties of senescent MtDNA are discussed with regard to the published properties of the rho- mutation in the yeast, S. cerevisiae.

Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K12

Abstract: A strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth was isolated from Escherichia coli K12 Genetic mapping and the molecular weight of the gene product suggest that the mutation is in the cou gene, specifying a sub-unit of DNA gyrase Nuclear organisation and segregation and placement of septa are grossly abnormal in the mutant at 42°C RNA synthesis and initiation of DNA replication are also affected at the restrictive temperature but the rate of DNA chain elongation continues almost undisturbed