Martha M. Howe

Partial correlation of the genetic and physical maps of bacteriophage Mu

Abstract λpMu phage particles that carry varying amounts of the Mu genome have been used to correlate the genetic and physical maps of phage Mu DNA. Two complementary techniques, restriction enzyme fragment analysis and electron microscope heteroduplex mapping, have been employed to identify the segments of Mu DNA present in the λpMu particles whose genetic complement had been determined by Mu amber mutant marker rescue. These analyses permitted us to assign a physical location of genes A to lys on one end (immunity end) of the Mu genome, and genes L to S on the other end (variable end). None of the hybrids described here included genes E to K which lie in the middle part of Mu DNA.

Correction and refinement of the genetic map of bacteriophage Mu.

Three hundred sixty-one amber mutations in essential genes of bacteriophage Mu have been assigned to 1 of 76 deletion groups on the basis of results of deletion mapping by marker rescue from 90 λpMu transducing phage. The original order of previously defined cistrons determined from prophage deletion mapping has been confirmed with the exception that cistron O has been deleted and cistron H has been found to map between cistrons E and F rather than between cistrons G and I. Mapping of mutations in the new cistrons T, U, V, W. and Y (Howe et al., 1979a, Virology 93, 303–319) has produced the following cistron order: ABClysDEHFGITJKLMYNPQVWRSU.

The S and U genes of bacteriophage Mu are located in the invertible G segment of Mu DNA

Abstract An F′ plasmid containing only the β and G segments of bacteriophage Mu DNA in a β-G-β structure was isolated as a LacZ− segregant of an F′lac plasmid containing Mu prophages inserted in the lacI and lacY genes. The segregant arose by homologous recombination between the similarly oriented G regions located in oppositely oriented Mu prophages whose α segments were directed toward the lacZ gene. Electron microscopic observation of single-stranded plasmid DNA from the segregant revealed the expected β-G-β stem and loop structure in which the double-stranded stem was the same length as, β and the single-stranded loop was the same length as G. Marker rescue experiments with Mu amber mutants defective in each of the known essential Mu genes showed that strains carrying this F′-β-G-β plasmid contained the wild-type alleles for S and U mutations but not for mutations in other genes. more detailed mapping of the R S U region by deletion mapping in Mu prophages and λpMu transducing phages produced two gene orders, R S U and R U S, thus indicating that the S and U genes were located in the invertible G segment rather than in β. Confirmation of this conclusion was obtained from results of DNA heteroduplexing experiments which showed that deletions ending within gene U end physically within G segment DNA. The location of essential genes S and U within G and the location and extent of G segment DNA which is nonessential for growth ( L. T. Chow, R. Kahmann, and D. Kamp, 1977 , J. Mol. Biol., 113, 591–609), taken together, reveal that the order of genes in viable Mu phage with G in the G(+) orientation is R S U.

Morphogenetic structures present in lysates of amber mutants of bacteriophage Mu.

The Mu phage particle is structurally similar to that of the T-even phages, consisting of an icosahedral head and contractile tail. This study continues an analysis of the morphogenesis of the Mu phage particle by defining the structural defects resulting from mutations in specific Mu genes. Defective lysates produced by induction of 55 amber mutants, representing 24 essential genes, were examined in the electron microscope and categorized into eight classes based on the observed phage-related structures. (1) Mutations in genes lys, F and G, and some H mutations, did not cause a visible alteration in particle structure. (2) Mutants defective in genes A, B, and C produced no detectable phage structures, consistent with their lack of production of late RNA. (3) Extracts defective in genes L, M, Y, N, P, Q, V, W, and R contained only head structures, and these appeared normal. (4) K-defective mutants accumulated free heads as well as free tails which were longer than normal and variable in length. (5) Tails which appeared normal were the only structures found in T- and some I-defective extracts. (6) Free tails and empty heads accumulated in D-, E-, and some I- and H-defective extracts. These heads were as much as 16% smaller than normal heads. The heads found in some I amber lysates had a protruding neck-like structure and unusually thick shells suggestive of a scaffolding-like structure. (7) Defects in gene J resulted in the accumulation of unattached tails and full heads. (8) Previous analysis of lysates produced by inversion-defective gin mutants fixed in the G(+) orientation demonstrated that S and U mutants produced particles lacking tail fibers (F.J. Grundy and M.M. Howe (1984), Virology 134, 296-317). In these experiments with Gin+ phages S and U mutants produced apparently normal phage particles. Presumably the tail fiber defects were masked by the production of S and U proteins by G(-) phages in the population.

Regulation and expression of the bacteriophage Mu mom gene: mapping of the transactivation (Dad) function to the C region

Expression of the bacteriophage Mu mom gene is under tight regulatory control. One of the factors required for mom gene expression is the trans-acting function (designated Dad) provided by another Mu gene. To facilitate studies on the signals mediating mom regulation, we have constructed a mom-lacZ fusion plasmid which synthesizes beta-galactosidase only when the Mu Dad transactivating function is provided. lambda pMu phages carrying different segments of the Mu genome have been assayed for their ability to transactivate beta-galactosidase expression by the fusion plasmid. The results of these analyses indicated that the Dad transactivation function is encoded between the leftmost EcoRI site and the lys gene of Mu; this region includes the C gene, which is required for expression of all Mu late genes. Cloning of an approx. 800-bp fragment containing the C gene produced a plasmid which could complement MuC- phages for growth and could transactivate the mom-lacZ fusion plasmid to produce beta-galactosidase. These results suggest that the C gene product mediates the Dad transactivation function.

Isolation of mutations defining five new cistrons essential for development of bacteriophage Mu

Abstract Three hundred new mutant strains of bacteriophage Mu with amber mutations in essential genes were isolated and characterized. Measurement of complementation between these new strains and strains carrying mutations in previously identified complementation groups revealed the existence of five new complementation groups: T , U , V , W , and Y . In general, mixed infection of cells by phage carrying mutations in different cistrons resulted in the production of a normal burst of 50 to 200 phage per cell; however, mixed infection with phage carrying mutations in certain pairs of adjacent cistrons gave a 10- to 20-fold lower burst of only 3 to 30 phage per cell. Cistron pairs which showed this reduced complementation were D - E , H - F , J - K , Y - N , Q - V , W - R , and S - U . In addition, mutations assigned to cistron I fell into three groups on the basis of their complementation behavior; phage carrying mutations in one group located at one end of the cistron showed weak complementation with phage carrying mutations in a second group at the opposite end of the cistron, but phage carrying mutations in the third group located between the first two groups showed no complementation with members of any of the three groups. The complementation analysis also showed that mutations previously assigned to separate complementation groups O and P really belong to a single complementation group P .

Kinetics and regulation of transcription of bacteriophage Mu

Mu transcription was analyzed by hybridization of [3H]uridine pulse-labeled RNA from heat-induced Mu lysogens to Mu DNA restriction fragments on nitrocellulose blots. Based on their time of appearance and dependence on Mu functions, we have defined three classes of transcripts: early, middle, and late. Replication-defective prophages containing A or B amber mutations or a deletion of the beta (right) end produced only early RNA derived from the left-most 8 to 10 kb of the Mu genome. A replication-proficient C amber mutant exhibited similar early transcription but at later times also produced middle transcripts from a region including C, which encodes the activator of late transcription. The C mutant did not produce late transcripts from the right-most 26 kb of the Mu genome encoding genes involved in phage morphogenesis and release. These results indicate that Mu DNA replication is required for efficient expression of middle RNA, which is itself required for expression of late transcripts. Amber mutations in essential genes other than A, B, and C had no significant effect on transcription except for polarity of one E mutation. Uninduced Mu c+ and Mu cts prophages produced very low levels of Mu-specific RNA derived from several regions including the c (immunity) gene and the region between genes B and C.

Cloning of DNA fragments of the right end of phage Mu and location of the HindIII, SalI, PstI, and BamHI restriction sites on the genetic map of Mu

Abstract The construction of three new plasmids containing the right, variable end of phage Mu DNA is described. They complete a collection of pKN plasmids derived by cloning specific restriction fragments of Mu DNA into plasmid cloning vehicles. The collection contains different length Mu DNA segments which encompass the entire phage genome. The Mu-specific genetic information of each cloned fragment was determined by marker rescue analysis of plasmid-containing strains with Mu amber mutant phages characterized by ODay et al. (K. ODay, D. W. Schultz, W. Ericsen, L. Rawluk, and M. M. Howe, 1979 , Virology 93 , 320–328). This analysis demonstrated the location of the Hin dIII, Sal I, Pst I, and Bam HI restriction sites on the genetic map of Mu and located the new essential genes V , W , and Y on specific restriction fragments of the physical map of Mu DNA. This plasmid bank has thus allowed a further refinement in the correlation of the genetic and physical maps of Mu DNA and should provide a useful source of specific DNA segments for further genetic and biochemical analysis of Mu functions.

Correlation of the genetic and physical maps in the central region of the bacteriophage Mu genome

Abstract DNA fragments resulting from cleavage of mature Mu DNA by the restriction endonuclease Eco R1 were cloned into a λ cloning vehicle, Charon4. Twelve clones containing genetic markers from the central region of Mu and eight clones containing markers from the right end of Mu were isolated. Restriction endonuclease and DNA heteroduplexing analyses of selected clones demonstrated that phages 4M121 and 4M136 contain the middle Eco R1 restriction fragment of Mu DNA cloned in opposite orientations within Charon4. The isolation and genetic mapping of deleted derivatives of these clones lacking varying amounts of Mu DNA were reported earlier [ODay, K., et al. (1979). Virology 93 , 320–328]. Four such deleted clones were analyzed by measuring single- and double-stranded DNA segments in heteroduplexes of deleted clone DNA and Mu DNA. The combination of these measurements and amber mutant marker rescue results allowed a precise localization of all 12 essential Mu cistrons in this region on the physical map of Mu DNA.

Location of the “variable end” of Mu DNA within the bacteriophage particle

Abstract When bacteriophage Mu is subjected to mild formaldehyde cross-linking conditions and spread for electron microscopy, the phage heads lyse and one end of the released DNA is found attached to the proximal end of the phage tail. If spreading is performed at pH 11.0–11.2 the DNA exhibits regions of partial denaturation in characteristic locations along the DNA molecule. Examination of the partial denaturation patterns of Mu DNA-tail complexes for wild-type Mu and for the insertion mutant MuX reveals that the tail is attached to a unique end of the DNA. Comparison of denaturation maps of DNA-tail complexes with those of Mu DNA heteroduplexes containing G bubbles and split ends indicates that the tail is attached to the variable end of Mu DNA. Similar analysis of denaturation maps of Mu DNA partially ejected through the tail demonstrates that the variable end is also the first end to be ejected from the phage particle. These results support the hypothesis that the variable end is the last section of Mu DNA to be packaged and the first to be released upon injection.