Morphogenesis of the tail fiber of bacteriophage T4

Summary A component of T₄ phage tail fiber was purified from the lysate of E. coli strain Bb infected with gene 35 defective mutant of T₄D (amB252). The purified component which occupies a part of the distal half fiber is formed under the control of genes 36, 37 and 38. The purified component was characterized and compared with the genes 35-36-37-38 directed half fiber. Although the components resembled each other, differences were observed in length, stability and chemical compositions. The results of a further decomposition of this component and the correlating characters of the gene 35 and 36 directed products were discussed.     

Analysis of sorbose resistance in Neurospora crassa in heterokaryons of sorbose resistant mutants; contribution to the genetics of active transport

Summary Suitable auxotrophic markers were introduced into sorbose-resistant mutants and the sorbose-sensitive wildtype strain. Pairwise combinations of one resistant and one sensitive strain each as well as of two sensitive strains were then grown on minimal-agar to obtain forced heterocaryons. The growth behaviour of these on minimal-agar with and without added sorbose was compared.Of seven resistant mutants, representing six separate genes, among which were genes A and B, six mutants were recessive to the wildtype. The seventh, representing gene C, was recessive only with regard to colony-size, but intermediate with regard to germination counts. Heterocaryons forced between pairs of 2 closely linked mutants (intragenic case of the type A^– ₁+A^– ₂) were resistant, as were the separate mutants. However two heterocaryons forced between pairs of unlinked mutants (intergenic case of the type A^–+B^–) were sorbose sensitive. Heterocaryons forced between A or B-mutants and the C-mutant mentioned, unlinked to either A or B (intergenic cases of the type A^–+C^– and B^–+C^–) were more sensitive than the separate mutants but more resistant than the wildtype.It follows that sorbose-resistant mutants in heterocaryons of the intergenic types can complement each others defects (no growth complementation), but can not do so in heterocaryons of the intragenic type. Their complementation is considered to be the result of the activity of the intact wildtype genes homologous to the defective ones that are contained together in the multinucleate cells of the heterocaryons. This complementation may be taken as evidence for the recessiveness resp. intermediate expression of the different resistant mutants.Since none of the mutants checked so far were dominant compared to the wildtype, none of them can be a regulator-mutant. The possibility of explaining them as suppressor mutants is restricted by their recessiveness to mechanisms of suppression giving a recessive phenotype. An alternative explanation suggests that the respective wildtype genes may contain structural information for the synthesis of permeases involved in sorbose transport. The mutants would then be resistant due to defective permeases. Their recessiveness is in full accord with this suggestion.

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II. Teil einer Habilitationsschrift bei der Naturwissenschaftlichen Fakultät der Universität München.     

The DNA-Delay Mutants of Bacteriophage T4

Mutants of phage T4 defective in genes 39, 52, 58-61, and 60 (the DNA delay or DD genes) are characterized by a delay in phage DNA synthesis during infection of a nonpermissive Escherichia coli host. Amber (am) mutants defective in these genes yield burst sizes varying from 30 to 110 at 37 C in E. coli lacking an am suppressor. It was found that when DD am mutants are grown on a non-permissive host at 25 C, rather than at 37 C, phage yield is reduced on the average 61-fold. At 25 C incorporation of labeled thymidine into phage DNA is also reduced to 3 to 10% of wild-type levels. Mutants defective in the DD genes were found to promote increased recombination as well as increased base substitution and addition-deletion mutation. These observations indicate that the products of the DD genes are necessary for normal DNA synthesis. The multiplication of the DD am mutants on an Su⁻ host at 37 C is about 50-fold inhibited if prior to infection the host cells were grown at 25 C. This suggests that a compensating host function allows multiplication of DD am mutants at 37 C in the Su⁻ host, and that this function is active in cells grown at 37 C prior to infection, but is inactive when the prior growth is at 25 C. Further results are described which suggest that the products of genes 52, 60, and 39 as well as a host product interact with each other.     

The dependence of HNO2 mutagenesis in phage T4 on ligase and the lack of dependence of 2AP mutagenesis on repair functions

Summary A temperature sensitive ligase allele of phage T4 reduced or eliminated HNO₂ induced reversion of am mutants. Since at the temperatures used, the ligase mutant is defective in the repair of some types of lethal lesions (i.e., UV, MMS and EMS induced lesions) these results indicate that HNO₂ mutagenesis may occur through a ligase dependent repair pathway. In contrast, 2AP induced mutation was not inhibited by mutants defective in the gene 30 ligase or in genes 32, 39, 41, 44, 45, 46, 47, 49, 52, 56, 58–61 and v. This indicates that 2AP mutagenesis probably does not depend on a repair pathway in phage T4.     

The genetics of dopa decarboxylase in Drosophila melanogaster

Of 204 mutations located in the 8–12 band Df(2L)130 region, 37B9-C1,2;37D1-2, 199 have been assigned to twelve lethal genes and one visible gene (hook). The 13 genes are not evenly distributed. Twelve, (possibly all thirteen) are in the seven band region 37B10-C4 giving a gene-to-band ratio of almost two. Only one gene, 1(2)37Cf, may be in the four band region 37C5-7, and none are localized in band 37D1. In situ hybridization places the dopa decarboxylase structural gene, Ddc, in or very close to band 37C1,2 (Hirsh and Davidson, 1981). The methyl dopa hypersensitive gene, 1(2) amd, is 0.002 map units distal to Ddc. Df(2L)VA17, 37C1,2; 37F5-38A1 may actually break in the 37C1,2 singlet. It places six genes, hook, 1(2)amd, and four lethal genes, in a maximum of five bands, 37B10, 11, 12, 13 and perhaps part of the 37C1,2 singlet and localizes six genes, Ddc plus five lethal genes, in a maximum of three bands; probably part of the 37C1,2 singlet plus bands, C3, and C4. Wild type activity of five of twelve lethal genes is necessary for female fertility. — Band 37C5 puffs at the time of pupariation; Puff Stages 8–10. Twelve of eighteen alleles of 1(2)37Cf havs been examined as heterozygotes over CyO and none affect the appearance of a homozygous 37C5 puff. — Of the 204 mutations considered here only one Ddc ^p1, affects the function of more than one gene. It eliminates Ddc ⁺ and l(2) 37Ca ⁺ function and at 30 ° C reduces l(2)37Ce ⁺ function. It is not a deficiency but could be a polar mutant.Prof. Beermann's co-authors are very pleased to dedicate this paper to him in honor of his sixtieth birthday and in recognition of his seminal, most significant, extensive, and authoritive contributions on the functional organization of chromosomes     

Supersensitive double mutants in yeast

Summary Double mutants containing the radiosensitive alleles uxsl (UVR^-EXR^-) and either uvsl-2 or uvsl 9-2 (both UVR^-EXR⁺) have been constructed. We find that these double mutants are extremely sensitive to UV: their LD₃₇ doses are 0.3–0.5 ergs/mm² as compared to 16–40 ergs/mm² for the radiosensitive parental strains carrying only a single mutant gene and 456 ergs/mm² for wild-type.     

Non-random distribution of transduction termini in transductants from the integrated R plasmid, R100-1

Summary Tra ⁺and tra ^–derivatives of drug resistance plasmid, R100-1, were isolated by phage P1 from an Hfr donor with integrated R100-1 and then analyzed by complementation tests with tra ^–point mutants of Flac. Tra ⁺derivatives of R100-1 carrying tetracycline resistance alone and those carrying all six drug-resistance genes could support transfer of tra ^–point mutants of Flac except Flac traJ, whereas all of tra ^–derivatives of R100-1 failed to complement any one of tra ^–point mutants of Flac. This suggests that these tra ^–derivatives of R100-1 carrying tetracycline resistance gene are deleted for all the transfer genes impaired in the Flac point mutants tested. We assume a hot point, probably a specific base sequence similar to an IS element, at the left of the tetracycline gene (Fig. 1) becomes a transduction terminus in transduction of the integrated R100-1 by phage P1. Complementation analysis of tra ^–derivatives carrying five resistance genes except the tetracycline gene led us to a supposition that a gene(s), probably analogous to traJ of the F plasmid, is located on R100-1 near the tetracycline gene which plays an important regulatory role for self-transfer as well as for the complementation of tra ^–Flac mutants.     

Functional significance of conserved residues in the phosphohydrolase module of Escherichia coli MutT protein

Escherichia coli MutT protein hydrolyzes 8-oxo-7,8-dihydro-2′-dGTP (8-oxo-dGTP) to the monophosphate, thus avoiding the incorporation of 8-oxo-7,8-dihydroguanine (8-oxo-G) into nascent DNA. Bacterial and mammalian homologs of MutT protein share the phosphohydrolase module (MutT: Gly37→Gly59). By saturation mutagenesis of conserved residues in the MutT module, four of the 10 conserved residues (Gly37, Gly38, Glu53 and Glu57) were revealed to be essential to suppress spontaneous A:T→C:G transversion mutation in a mutT^– mutator strain. For the other six residues (Lys39, Glu44, Thr45, Arg52, Glu56 and Gly59), many positive mutants which can suppress the spontaneous mutation were obtained; however, all of the positive mutants for Glu44 and Arg52 either partially or inefficiently suppressed the mutation, indicating that these two residues are also important for MutT function. Several positive mutants for Lys39, Thr45, Glu56 and Gly59 efficiently decreased the elevated spontaneous mutation rate, as seen with the wild-type, hence, these four residues are non-essential for MutT function. As Lys38 and Glu55 in human MTH1, corresponding to the non-essential residues Lys39 and Glu56 in MutT, could not be replaced by any other residue without loss of function, different structural features between the two modules of MTH1 and MutT proteins are evident.     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Conditional lysis ofEscherichia coli by the fusion of extracellular (STA) to periplasmic (LTB) enterotoxins: Apparent phenotypic suppression of lactose permease

The expression of a methanol-soluble, heat-stable enterotoxin (ST_A) fused to the B subunit of the heat-labile enterotoxin (LT_B) at 35°C or higher temperatures caused strains ofEscherichia coli deficient in lactose permease to behave on indicator media as Lac⁺; however, at 33°C or lower temperatures the original Lac^– phenotype of the host strains was maintained. The apparent phenotypic suppression oflacY was shown to be due to lysis of a fraction of the bacteria and the consequent release of active-galactosidase to the culture supernatant. After incubation at 37°C for 1 h, the cultures were committed to lyse. Plasmid and chromosomal mutants that do not show this phenotype were isolated by selecting Lac^– colonies at the unpermissive temperature. The mutations on the plasmids were localized in both the heat-stable and the heat-labile enterotoxin genes. Chromosomal mutants that show normal levels of-galactosidase and fused toxins have also been isolated.     

Relaxation of supercoiled DNA associated with induction of heat shock proteins in Escherichia coli

Bacillus subtilis 168 is known to possess two thymidylate synthase (TSase; EC 2.1.1.45) genes: thyA and thyB. thyB encodes a thermosensitive TSase (inactivated at 46° C) which, in wild-type cells, accounts for only 5–8% of the total cellular TSase activity. In order to investigate the thermal lability of TSaseB we have analyzed the thyB genes of B. subtilis 168 and of an unrelated strain B. subtilis ATCC6633, which is shown here to have a temperature-resistant TSaseB. This conclusion is supported by the frequency of appearance of spontaneous Thy^– mutants at 37° C and 46° C, and by the analysis of clones containing the thyB genes from the two strains. The nucleotide sequences of these two thyB genes were compared.     

Minimal CK2 activity required for yeast growth

Protein kinase CK2 is essential for the growth of Saccharomyces cerevisiae. Yeast cells that lack the functional genes coding for both the catalytic subunits of protein kinase CK2 can grow only if they are complemented by exogenous cDNAs coding for this subunit. A series of deletion mutants of CK2α from Xenopus laevis was constructed. These mutants that have carboxyl end deletions yield a CK2α product that varies over four orders of magnitude in its capacity to phosphorylate casein in vitro. Complementation of yeast RPG41-1a, a mutant defective in CKA1 and CKA2 genes, with wild-type X. laevis CK2α and with cDNAs coding for truncated CK2α having amino acids 1–328 and 1–327 resulted in cells that grew in gal-minimal media at 30 ^∘C as well as the cells harboring the yeast CKA2 gene. However, the growth was significantly diminished when cells were complemented with X. laevis CK2α containing 1–326 amido acids. This mutant has 0.6% of the catalytic activity of the wild-type enzyme. Yeast cells that expressed CK2α 1–324 and 1–323 which have 10-and 100-fold less activity, respectively, were not able to grow. The growth of cells containing the CK2α 1–326 mutant was very sensitive to temperature, and minimal growth was observed at 37 ^∘C. This mutant was also more sensitive to UV radiation but was not significantly affected by 0.4 M NaCl.Both authors contributed equally to this work     

Gene Expression and Stability of mRNA Affected by DNA-Arrested Synthesis in Gene 59, 46, and 47 Mutants of Bacteriophage T4

The effect of bacteriophage T4 gene 59 mutations (DNA-arrested synthesis) on kinetics of DNA synthesis, gene expression, and stability of mRNA has been studied. When Escherichia coli B was infected by a T4 gene 59 mutant, DNA synthesis proceeded to increase linearly after initiation, but started to decrease at 8 min and was completely arrested at 12 min at 37°C. At various incubation temperatures (20 to 42°C), the initial rates and times of arrest of DNA synthesis were different, but the total amount of DNA synthesized was constant. This result supports the hypothesis that function of gene 59 is required for the conversion of 63S DNA molecules to other replicative intermediates (39). The abnormality in protein synthesis caused by gene 59 mutation is manifested by (i) a delayed shutoff in the expression of early proteins (gene 43, 46, 39, 52, 63, 42-45, and some unidentified proteins), (ii) a reduced rate of late gene expression (gene 34, 37, 18, 20, 23, wac, 24, 22, 38, and 19), and (iii) an absence of cleavage of certain late proteins (23, 24, IPIII and 22 to 23^*, 24^*, IPIII^*, and small fragments). It appears that there was no effect on the expression of gene 33, 55, and 32 by a mutation in gene 59. Results obtained from an addition of rifampin at the prereplicative cycle after infection indicated that mRNA from genes 43, rIIA, 46, 39, 52, and 63 are more stable in T4amC5 (gene 59) than in wild-type-infected cells. mRNA remained functional longer in mutant-infected cells, and this may explain the prolonged synthesis of certain early proteins. The gene expression of other DNA arrested mutants—those in genes 46 and 47—showed a pattern of abnormal protein synthesis similar to that found in gene 59 mutant-infected cells, except more late proteins are synthesized. The gene expression in terms of phage DNA structure is discussed.     

Isolation and characterization of five genotypic mutants of chlorate-resistant cyanobacteria unable to utilize nitrate

Spontaneous mutants of the cyanobacteriumSynechococcus PCC 7002 resistant to chlorate were isolated. Either 40mM or 400mM Na₂ClO₃ was used as the selective agent. Putative Chl^r colonies were picked onto medium containing ammonia as the sole N source, then replicaplated to media containing either NH₄ ⁺, NO₂ ^– as N sources. Of 252 putative mutants, 106 were able to use either NH₄Cl or NaNO₂ but not NaNO₃ as their sole source of nitrogen. All of the mutant isolates had generation times similar to wild-type 7002 when grown on either ammonium (3.8–4.1 h/generation) or nitrite (4.5–4.7 h/generation). None had detectable methyl viologensupported nitrate reductase activity and are thus phenotypically NRase^–. The Chl^r mutants had photomediated O₂ production and dark O₂ uptake rates similar to the wild type and responded similarly to selected metabolic inhibitors. They expressed increased levels of phycocyanin (PC) synthesis under normal, nitrogen-replete growth conditions, but rapidly lapsed into a chlorotic state upon a shift to either medium containing nitrate or to N-free medium. Genetic analysis of the Chl⁴ mutants indicated that each could be rescued by direct transformation with chromosomally derived DNA from the wild-type strain. Frequencies of transformation for the mutants were characteristic for single genetic lesions in this cyanobacterium. On the basis of marker rescue by a cosmid library of wild-type DNA, the NRase^– mutants could be grouped into five distinctive genotypic families.     

Isolation of Rec? mutants from an F-prime merodiploid strain of Escherichia coli K-12

Summary This paper describes a method of screening mutagenised populations of an E. coli gal _A ^– gal _B ^– F-prime merodiploid for mutants defective in recombination. The method relies on scoring colonies on Eosin-Methylene Blue agar that have fewer than normal numbers of Gal⁺ papillae. With a suitable choice of gal ^–mutations most of the papillae arise by recombination and some of those colonies with less than normal numbers prove to be defective in some aspect of recombination or DNA repair. In addition to strains carrying mutations that can be ascribed to known loci, several novel mutant phenotypes were identified.     

Restriction/modification in Streptococcus thermophilus: isolation and characterization of a type II restriction endonuclease Sth455I

Streptococcus thermophilus strain CNRZ 455 produces a type II restriction endonuclease designated Sth455I. This enzyme was isolated from cell extracts by anionic and cationic exchange chromatography. This yielded an enzyme preparation free of non-specific nucleases. The optimal reaction conditions for Sth455I are: MgCL₂, 30 mm; pH range, 8–9; incubation temperature, 37–42°C; and a high NaCl concentration, 100–200 mm. The results of single- and double-digestion experiments indicates that Sth455I is an isoschizomer of BstNI and EcoRII showing different sensitivity to methylation. The enzyme exhibits restriction activity on the DNA of three bacteriophages of S. thermophilus and no activity on the phage lytic for strain CNRZ 455. The restriction/modification system associated with this strain is discussed.     

Properties of two bacteriocins synthesized byLeuconostoc strains

Mesenterocin 52 (Mes-52), was produced byLeuconostoc mesenteroides ssp.mesenteroides strain FR52 and dextranicin 24 (Dex-24) byLeuconostoc mesenteroides ssp.dextranicum strain J24. Dex-24 had a very narrow spectrum of antibacterial activity: it inhibited only some other strains ofLeuconostoc, contrary to Mes-52, which antagonized alsoListeria andEnterococcus sp. Mes-52 was partially purified by ammonium sulfate precipitation, gel filtration, cation exchange, and hydrophobic interaction chromatography. Mes-52 was biosynthesized and excreted into the medium during growth phase. At different temperatures media, there was an inverse relationship between the final bacteriocin-specific productivity and the mean growth rate. After novobiocin treatment, different mutants of strain FR 52 were obtained: nonproducing-sensitive (Bac^–Imm^–) and nonproducingimmune (Bac^–Imm⁺) strains. Many changes in the plasmid profiles of several mutants were detected by electrophoresis.