Relationships among genes and gene products of bacteriophage BF23

Twenty-five gene products of bacteriophage BF23 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functions were studied in relation to type I and II genes classified by means of genetic complementation tests. All the type I mutants were defective in the synthesis of a tail protein, L3. In addition, 4 type I gene products, L5 (gp21), L7 (gp20), L8 (gp29), and L9 (gp25), were identified as constituents of tails (gp21 denotes that a protein is a product of gene 21). Three type IIb mutants in genes 10, 14, and 19 diminished substantially the production of late proteins, including tail and head proteins, and the two other type IIb mutants in genes 1 and 2 were defective in the synthesis of both early and late proteins. Of 14 type IIa mutants, at least 6 were defective in phage DNA synthesis and 2 were defective in the synthesis of head proteins. The defect in the head donor activities of type IIa mutants in extract complementation tests was due to the failure of the formation of mature heads containing DNA. The above results support directly the results of the genetic characterization of BF23 genes.     

Elbow extensor muscles of the horse: postural and dynamic implications.

Based on histochemical and immunohistochemical evidence, horse elbow extensor muscles are composed of two morphologically distinct muscle groups. The long and lateral heads of the triceps brachii are large, predominantly type II (presumed fast) muscles. The long and lateral heads of the triceps together account for 96% of the weight of the elbow extensors (long head of triceps is 81%). The long and lateral heads contain three histochemical fiber types: types I, IIa and IIb. Type I muscle fibers account for approximately 18 and 27% of the fibers in the long and lateral heads of the triceps, respectively. In the lateral head, type IIa and IIb fibers account equally for the remaining 70%, while in the long head of the triceps type IIb fibers predominate (50%) over type IIa fibers (32%). In contrast, the much smaller medial head of the triceps (2% of triceps mass) and the anconeus (2% of mass) contain almost exclusively type I muscle fibers. It is hypothesized that the medial head and anconeus, with their slow fibers, contribute to the postural maintenance of the forelimb by preventing flexion at the elbow joint during passive stance. The larger long and lateral heads, with their generally fast fiber populations, are most likely important during dynamic activity.     

Isolation and characterization of temperature-sensitive mutants of Sendai virus

Sixteen temperature-sensitive mutants of Sendai virus were isolated from mutagenized stocks (10 mutants, designated numerically) and persistently infected cultures (6 mutants, designated alphabetically). Based on complementation tests, virion-associated activities, thermal inactivation, and viral RNA and hemadsorbing antigen synthesis as well as virion production in chick lung embryo cells at nonpermissive temperature, these mutants were divided into seven groups as follows. i) HANA group mutants (ts-5, -9, -10, -201), defective in hemagglutinin-neuraminidase protein, complementation group I. ii) F group mutants (ts-18, -108), defective in hemolytic and cell-fusing activity, complementation group II. iii) Ts-43, defective in RNA polymerase activity, complementation group III. iv) Ts-23, defective in RNA polymerase activity, interfered with the other mutants in complementation tests. v) Ts-25, defective in the incorporation of hemagglutinin-neuraminidase protein into the virion at the stage of virus assembly. vi) Ts-110, belongs to F group mutants on one hand, but is considered to carry another undetermined defect. vii) C group (carrier culture-borne group) mutants (ts-a, -b, -c, -d, -e, -f), defective lesion not yet determined and belong to neither complementation group I nor II. Assignment of mutants in groups iv), v), vi), and vii) to complementation groups could not be achieved.     

Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization.

We have isolated mutants defective in DNA topoisomerases and an endonuclease from the fission yeast Schizosaccharomyces pombe by screening individual extracts of mutagenized cells. Two type I topoisomerase mutants (top1) and three endonuclease mutants (end1) were all viable. The double mutant top1 end1 was also viable and, in its extract, Mg2+- and ATP- dependent type II activity could be detected. Three temperature-sensitive (ts-) mutants having heat-sensitive (hs-) type II enzymes were isolated, and the ts- marker cosegregated with the hs- type II activity. All the ts- mutations fell in one gene (top2) tightly linked to leul in chromosome II. The nuclear division of single top2 mutants was blocked at the restrictive temperature, but the formation of a septum was not inhibited so that the nucleus was cut across with the cell plate. In contrast, the double top1 top2 mutants were rapidly arrested at various stages of the cell cycle, showing a strikingly altered nuclear chromatin region. The type II topoisomerase may have an essential role in the compaction and/or segregation of chromosomes during the nuclear division but also complement the defect of the type I enzyme whose major function is the maintenance of chromatin organization throughout the cell cycle.     

Recombination in amber-mutants of bacteriophage T4B. I. Recombination and complementation in phage T4 amber-mutants]

In studying intergenic and intragenic complementation in amber mutants in genes of phage T4 controlling the synthesis of phage tail fibres the data have been obtained indicating the dependency of the results of complementation tests on those of crosses of respective markers. The results obtained show that in complementation of amber mutants of phage T4 the phage yield varies widely and depends on the location of markers on the phage genetic map.     

Studies on the In Vitro Assembly of Bacteriophage φ80 and φ80-Lambda Hybrids

Suppressor-sensitive (sus) mutants of bacteriophage 80 defective in late functions were classified, by means of in vitro assembly tests, into two complementation groups: head donors and tail donors. Each group of mutants was subdivided, by means of two-factor crosses, into six cistrons. Deletion mapping revealed clustering of tail and also of head cistrons. The two clusters were located in the left arm of vegetative 80 (the tail specifying cluster being distal). In vitro cross complementation between 80 and lambda sus mutants revealed that whereas lambda heads could quite efficiently bind 80 tails to form viable phage, the union of 80 heads and lambda tails was very much less efficient. Deletion mapping of the 80 sus mutants, using both 80 and i⁸⁰h^λ deletion lysogens indicated congruent gross gene arrangement in the two related bacteriophages.     

Electron microscopy of virulent phages for Streptococcus lactis.

Electron microscopic studies were made on eight virulent Streptococcus lactis bacteriophages. These phages were taken as representative of eight host range groups established in a study of 75 phage isolates and 253 hosts (213 S. lactis, 22 S. cremoris, 18 S. diacetilactis). The phages studied were shown to have an isometric hexagonal head and noncontractile tails, usually several times longer than the head diameter. The virus heads were octahedral. The phages investigated represented three morphological types on the basis of head diameter , tail thickness, and tail length. These dimensions were approximately: for type I phages, 63, 172, and 11 nm, respectively; type II, 73, 200, and 20 nm, respectively; and type III, represented here by a single phage, 98, 551, and 12 nm, respectively. The tail surface revealed a different arrangment of the structural subunits which lent a helical appearance to the tails of type I and II phages and a guaffered tube appearance to the tail of type III phage. The number of turns along the tail axis, turn length, axial pitch, and helix angle were: type I, 32, 12 to 13 nm, 7.14 nm, and 11 degrees 43', respectively; type II, 24, 24, to 28 nm, 40.00 nm, and 32 degrees 30', respectively; and type III, 120, 12 nm, and no visible slope towards the axis. The morphology types showed complete correlation with serological groups, but not with groups based on host range pattern.     

Transduction of plasmid antibiotic resistance determinants with pseudo-T-even bacteriophages

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 from Escherichia coli recA(+)- and recA(-)-donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc segE uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages lambda, T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limited in vivo by modification-restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification-restriction systems EcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification-restriction system.     

Formate-nitrate respiration in Salmonella typhimurium: studies of two rha-linked fdn genes

Localized mutagenesis was used to obtain rha-linked mutations in Salmonella typhimurium, resulting in defects in the nitrate reductase-linked formate dehydrogenase (FDHN). The fdn mutants obtained fell into two groups which differed in several respects. Group I isolates lacked FDHN activity under all conditions examined and exhibited wild-type levels of the hydrogenase-linked formate dehydrogenase (FDHH). Group II isolates appeared defective in FDHN only when freshly prepared extracts were assayed; restoration of both FDHN and formate-nitrate reduction activity occurred on incubation of extracts for 2 to 3 h. Protease inhibitors prevented restoration. Group II isolates were also characterized by a conditional FDHH activity; this activity was absent unless the growth medium designed to optimize wild-type FDHH was altered either by lowering glucose concentration or by adding thiosulfate. Cotransduction of fdn with rha ranged from 4 to 22% for the group I isolates and from 20 to 40% for the group II isolates. Temperature-sensitive isolates from both groups synthesized FDHN activity with altered thermostability. In vitro complementation occurred in mixed extracts of amber mutants of the two respective classes. The results are consistent with two distinct rha-linked fdn genes, for which we suggest using the designations fdnB (group I) and fdnC (group II).     

Genetic mapping of regA mutants of bacteriophage T4D.

SP62, a mutant of bacteriophage T4 shown by Wiberg et al. (1973) to be defective in regulation of T4 protein synthesis, was shown by complementation tests to define a new gene, regA, and by intergenic mapping to lie between genes 43 and 62. The mapping involved crossing SP62 with a quadruple amber mutant defective in genes 42, 43, 62, and 44, selecting all six classes of amber-containing recombinants caused by single crossover events, and then scoring the presence or absence of SP62 in these recombinants. In addition, 15 new, spontaneous regA mutants were isolated, and 13 of these were mapped against each other; a total of eight different mutation sites were thus defined. Most of the new mutants were isolated as pseudorevertants of a leaky amber mutant in gene 62, according to Karam and Bowles (1974), whereas one was identified by virtue of the "white ring" around its plaque, a phenotype possessed by all the regA mutants at high temperature, SP62 was renamed regA1, and the new mutants were named regA2, regA3, etc.     

Diaphragm capillarity and oxidative capacity during postnatal development.

In the cat diaphragm, fiber capillarity, cross-sectional area, and succinate dehydrogenase (SDH) activity were measured across the first 6 wk of postnatal development. Fibers were classified as type I, IIa, IIb, or IIc on the basis of staining for myofibrillar adenosinetriphosphatase (ATPase). Capillaries were identified in sections stained for ATPase at pH 4.2. Fiber cross-sectional areas and SDH activities were quantified using an image-processing system. During postnatal development, the proportions of type I fibers increased while type II fibers decreased. At birth, all type II fibers were IIc. From the 1st to the 2nd postnatal wk, the proportion of type IIc fibers decreased while the numbers of IIa and IIb increased. Thereafter the proportion of type IIb fibers continued to increase while the number of IIa steadily declined. At birth, capillarity, cross-sectional areas, and SDH activities of type I and II fibers were low compared with other postnatal age groups. Fiber cross-sectional areas increased progressively with age. The number of capillaries surrounding type I and II fibers increased markedly by the 2nd wk and then continued to increase at a slower rate. The number of capillaries per fiber area reached a peak by the 2nd wk and then declined as fiber cross-sectional area increased. Postnatal changes in capillarity depended on fiber type, being greatest in IIb. SDH activities of type I and II fibers were initially low during the first 2 postnatal wk and then peaked by the 3rd wk. After the 6th wk, fiber SDH activities decreased to adult values. Among the type II fibers, IIb showed the greatest change in SDH activity during early postnatal development.     

CAENORHABDITIS ELEGANS Deficiency Mapping

Six schemes were used to identify 80 independent recessive lethal deficiencies of linkage group (LG) II following X-ray treatment of the nematode Caenorhabditis elegans. Complementation tests between the deficiencies and ethyl methanesulfonate-induced recessive visible, lethal and sterile mutations and between different deficiencies were used to characterize the extents of the deficiencies. Deficiency endpoints thus helped to order 36 sites within a region representing about half of the loci on LG II and extending over about 5 map units. New mutations occurring in this region can be assigned to particular segments of the map by complementation tests against a small number of deficiencies; this facilitates the assignment of single-site mutations to particular genes, as we illustrate. Five sperm-defective and five oocyte-defective LG II sterile mutants were identified and mapped. Certain deficiency-by-deficiency complementation tests allowed us to suggest that the phenotypes of null mutations at two loci represented by visible alleles are wild type and that null mutations at a third locus confer a visible phenotype. A segment of LG II that is about 12 map units long and largely devoid of identified loci seems to be greatly favored for crossing over.     

Morphogenesis of Bacteriophage φ80: Identification of the Cistron 13 Product

An in vitro complementation reaction leading to the assembly of bacteriophage phi80 tails from component proteins is described. Tail assembly occurs when a lysate of any mutant in cistron 13 is mixed with a second lysate of a mutant in any of the other cistrons involved in tail formation. Lysates of mutants that are blocked in tail formation contain phage heads that can unite with free tails to form infective particles. The rate of the complementation reaction shows little dependence upon temperature, suggesting that the assembly depends largely upon the kinetic encounter of the interacting components. The tail component missing in cistron 13 mutant lysates was purified approximately 55-fold and shown to be, at least in part, a protein having a molecular weight of approximately 22,000. This protein was also released from highly purified infective phi80 particles after osmotic shock followed by heattreatment, suggesting that it most probably is an integral structural protein of the phage tail. Lysates of mutants of bacteriophage lambda that are defective in tail formation were shown to contain a tail component identical with or similar to the phi80 cistron 13 product.     

T-even-type bacteriophages use an adhesin for recognition of cellular receptors

Protein 38 of the Escherichia coli phage T4 is thought to be required catalytically for the assembly of the long tail fibers of this phage. It is shown that this protein of phage T2 and the T-even-type phage K3 and Ox2 act differently. It was found that NH2-terminal fragments of the protein, expressed from cloned fragments of gene 38 of phage K3, bind to gene 38 amber mutants of phage T2. Such phage or T2 gene 38 amber mutants, grown on a non-permissive host, possess a complete set of six tail fibers but are non-infectious. Both types of non-infectious phage could be repaired by incubation with an extract of cells harboring a cloned gene 38 of a host range mutant of phage K3, K3hx. The repaired phages had the host range of K3hx and not of T2. Immuno-electron microscopy showed that protein 38 is located at the free ends of the long tail fibers of phages T2, K3 and Ox2. The protein serves the recognition of the cellular receptor, i.e. it acts as an adhesin.     

Genetic studies of coliphage 186. I. Genes associated with phage morphogenesis.

In coliphage 186, 22 essential genes were defined by complementation studies with amber mutants. Eighteen genes were associated with phage morphogenesis: 11 with phage tail formation, and 7 with phage head formation. The remaining four genes are discussed in the accompanying paper (S. M. Hocking and J. B. Egan, J. Virol. 44:1068-1071, 1982).     

Studies on plasmid replication. III. Isolation and characterization of replication-defective mutants

Some 85 Staphylococcus aureus mutants phenotypically thermosensitive for penicillinase plasmid segregation (Seg-) have been isolated and characterized. Some of the mutations were plasmid-linked and those studied in detail were found to be defective in plasmid replication, most problbly at the initiation stage. Analysis of the segregation behavior of these mutants suggested a figure of 2.7 for the average number of plasmid copies per cell in a random culture. Other mutations were host chromosome-linked and these could be divided into at least three classes on the basis of their ability to maintain plasmids of the two different incompatibility sets: some were defective for type I plasmids, some for type II, and some for both types. One host mutant, defective in segregation of type I but not type II plasmids, was defective in polymerization of both.     

Biochemistry of DNA-defective mutants of bacteriophage T4. VI. Biological functions of gene 42.

Bacteriophage T4 gene 1 and 42 amber mutants (defective in deoxynucleoside monophosphate kinase and deoxycytidylate hydroxymethylase, respectively) are able to synthesize DNA in cell-free lysates prepared as described by Barry and Alberts (1972), in contrast to their inabliity to do so in plasmolyzed and toluenized cell systems. Addition of extracts containing an active gene 1 or 42 product has no effect on synthesis in lysates defective in the respective gene. Thus, if these enzymes do play additional direct roles in replication, these roles are not manifest in the lysed-cell system. The gene 42 mutant am N122/m, a double mutant bearing an additional defect in DNA polymerase, is unable to synthesize DNA in these lysates. This inability is overcome by addition of extracts containing an active T4 DNA polymerase. m is a leaky amber mutation which reduces DNA polymerase activity to a very low level. However, this level is high enough to allow positive genetic complementation tests with gene 43 mutants. Two other gene 42 amber mutants contain additional defects: am 269 induces only half the normal level of DNA polymerase, and am C87 fails to induce a detectable level of thymidylate synthetase. These defects do not result from pleiotropic effects of the gene 42 mutations. In plasmolyzed cells, temperature-sensitive gene 42 mutants fail to synthesize DNA under conditions where replication forks and 5-hydroxymethyl-dCTP are present. This supports the idea that the gene 42 protein is directly involved in DNA synthesis.     

Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae. Characterization of mutants in 34 complementation groups

To identify nuclear functions required for cytochrome c oxidase biogenesis in yeast, recessive nuclear mutants that are deficient in cytochrome c oxidase were characterized. In complementation studies, 55 independently isolated mutants were placed into 34 complementation groups. Analysis of the content of cytochrome c oxidase subunits in each mutant permitted the definition of three phenotypic classes. One class contains three complementation groups whose strains carry mutations in the COX4, COX5a, or COX9 genes. These genes encode subunits IV, Va, and VIIa of cytochrome c oxidase, respectively. Mutations in each of these structural genes appear to affect the levels of the other eight subunits, albeit in different ways. A second class contains nuclear mutants that are defective in synthesis of a specific mitochondrial-encoded cytochrome c oxidase subunit (I, II, or III) or in both cytochrome c oxidase subunit I and apocytochrome b. These mutants fall into 17 complementation groups. The third class is represented by mutants in 14 complementation groups. These strains contain near normal amounts of all cytochrome c oxidase subunits examined and therefore are likely to be defective at some step in holoenzyme assembly. The large number of complementation groups represented by the second and third phenotypic classes suggest that both the expression of the structural genes encoding the nine polypeptide subunits of cytochrome c oxidase and the assembly of these subunits into a functional holoenzyme require the products of many nuclear genes.     

Genetic complementation analysis of Escherichia coli type 1 somatic pilus mutants.

A genetic complementation analysis of 75 stable nonpiliated mutants of a type 1 piliated strain of Escherichia coli K-12, AW405, was performed. Strains containing pairs of pil mutations were constructed by the infectious transfer of an F101 plasmid containing one pil mutation into E. coli K-12 AW 405 containing another pil mutation. The presence or absence of type 1 pili on the merodiploid strains was determined by agglutination with type 1 pilus antiserum. All 75 mutants fell into one of four complementation groups. The pattern of complementation defined three cistrons involved in pilus formation, pilA, pilB, and pilC. The fourth complementation group was composed of a large number of mutants defective in both pilA and pilB functions.