No description available.

No description available.

The conversion of raw forage crops such as grass and cereals into silage is brought about by a fermentation carried out by the indigenous lactic acid bacteria. The most important of the lactic acid bacteria in terms of silage fermentations are members of the general <i>Lactobacillus</i> and <i>Pediococcus</i>. Due to recent advances in the genetics of the lactic acid bacteria, there is now significant potential to modify strains by genetic manipulation and hence create a new range of strains with additional desirable characteristics such as cellulase production which can be used as silage additives. The strains used in this study were all natural silage isolates and were identified as either <i>Lactobacillus plantarum</i> or <i>Pediococcus pentosaceus</i> using a range of characteristics including homo/heterofermentative activity, Gram staining, the API 50CH system and growth temperature. Six strains of <i>L. plantarum</i> and twelve strains of <i>P. pentosaceus</i> were isolated. All were screened for the presence of native plasmids, with a range of sizes from two kilobases (kb) to eleven kb identified within the <i>L. plantarum</i> strains and a more limited size range identified within the <i>P. pentosaceus</i> strains. None of the ioslates were naturally transformable so electroporation was used as a means to facilitate the uptake of exogenous DNA. The transformation protocol was optimised by examining the effects of electroporation buffer, growth conditions of the bacteria, DNA concentration and various electrical parameters. The optimised procedure used exponential phase cells, 0.5M sucrose as the electroporation solution, one μg plasmid DNA, electrical parameters of 2.5 kilovolts, 100 ohms resistance and 25 microFarads capacitance followed by an expression period of two hours.

Physarum polycephalum is a Myxomycete or true slime mould, the life cycle of which alternates between two growing phases; amoebae and plasmodia. Strains of the Colonia isolate of Physarum polycephalum form plasmodia within single clones of amoebae; this is in contrast to strains of other isolates which require the sexual fusion of two amoebae of different mating type for plasmodium formation to occur. The mode of plasmodium formation by Colonia strains enables them to be used for the isolation of mutant plasmodia after anoebal mutagenssis and direct sereening of plasmodia. This thesis reports the derivation of a Colonia strain, CL, (Colonia Leicester) for mutant isolation purposes together with the construction of isogenic strains of heterothallic mating type. These strains have been used to isolate and genetically analyse strains carrying auxotrophic mutations which can only be tested for in the plasmodial stage. Attempts have also been made to devise selective techniques for the isolation of strains carrying specific classes of mutation, in particular a strain deficient in thymidylate synthetase activity. A series of investigations employing microdensitometric estimation of nuclear DNA content, time-lapse microcinematography and genetic methods has indicated that plasmodium formation by Colonia amoebae is probably apogamous and not homothallic as was previously reported by other workers.

No description available.

This work describes the identification of a <i>Schizosaccharomyces pombe</i> homologue of the <i>Saccharomyces cerevisiae CDC37 </i>gene. Homologues have been previously identified in a variety of organisms, including Drosophila, chicken, mouse, rat and human. The <i>S. pombe </i>gene has been named <i>cdc37⁺</i> and encodes a protein with a predicted molecular weight of 52 kDa. The protein shows extremely high sequence similarity with Cdc37p from <i>S. cerevisiae</i> for the first 40 amino acids at the N-terminus but, as with the homologues from other species, this similarity is considerably less over the remainder of the protein. Deletion of <i>cdc37⁺</i> results in a lethal phenotype, establishing that the gene is essential for viability. The null mutant could survive when <i>cdc37⁺</i> was expressed from the thiamine-regulatable low strength expression vector pREP81; when expression was repressed, the cells showed a variety of phenotypes, becoming swollen, misshapen and/or elongated. The diversity of phenotypes observed suggests that Cdc37 has several biological roles. Elongation of <i>S. pombe</i> cells indicates a cell cycle defect and measurements of cell length during Cdc37 depletion confirmed significant elongation in <i>Dcdc37</i> cells when <i>cdc37⁺</i> expression from the plasmid pREP81 was repressed. FACS and cytological analysis indicated that cell cycle defects occurred in both the G2 and mitotic phase of the cell cycle. Genetic analysis carried out to investigate the mechanism of cell cycle arrest suggests that Cdc37 may be involved with the Cdc2/Cdc13 complex, which controls entry into mitosis from the G2 phase, <i>cdc37⁺</i> was strongly overexpressed in wild-type cells from the high strength expression vector pREP1 with no effect. However, in strains carrying either of two <i>cdc13^ts</i> alleles, strong <i>cdc37⁺</i> overexpression severely reduced in restrictive temperature. A similar but more modest effect was seen in strains carrying various <i>cdc2^ts</i> alleles. Preliminary biochemical studies were also carried out, which showed that the Cdc2 protein level was lower than normal in cells depleted for Cdc37.

No description available.

Control over the mitotic cell division cycle of the fission yeast <I>Schizosaccharomyces pombe</I> is exerted at two main points, mitotic entry and an earlier point, situated prior to DNA replication, known as Start. At Start, cells become committed to one of a number of developmental pathways. The genes <I>cdc2</I><SUP>+</SUP> and <I>cdc10</I><SUP>+</SUP> are both required for commitment to the mitotic pathway. The <I>cdc22</I><SUP>+</SUP> gene encodes an enzyme which functions in S phase, and is periodically expressed at the G1/S boundary. Cdc10 binds elements in the <I>cdc22</I><SUP>+</SUP> promoter known as MCBs, and synthetic MCBs, inserted upstream of a reporter gene, confer periodicity of expression upon it. A second reporter gene has been constructed, under the control of sequences from the <I>cdc22</I><SUP>+</SUP> promoter, and this has been used, together with the MCB-controlled reporter, to investigate the regulation of <I>cdc22</I><SUP>+</SUP> under various conditions of cell cycle arrest. It has been shown that <I>cdc2</I><SUP>+</SUP> is not required at Start for MCB-mediated gene expression. In <I>cdc10</I><SUP>ts</SUP> arrested cells, <I>cdc22</I><SUP>+</SUP>, and the reporter gene under the control of the <I>cdc22</I><SUP>+</SUP> promoter, are both expressed. In contrast, the reporter gene under only MCB control is not expressed independently of <I>cdc10</I><SUP>+</SUP>. This suggests the existence of at least one <I>cis</I>-acting element within the <I>cdc22</I><SUP>+</SUP> promoter, additional to MCBs and required for <I>cdc10</I><SUP>+</SUP>-independent gene expression. The roles of the two clusters of MCBs that are found within the <I>cdc22</I><SUP>+</SUP> promoter have also been investigated.

No description available.

One way to approach the question of the evolution of bacteria and bacteriophages is by examining their DNA sequence homology. The electron microscope is a tool that allows the detection and measurement of regions of single- and double-stranded DNA. By using the techniques of DNA heteroduplex formation and DNA spreading I was able to use the electron microscope to seek regions of DNA sequence homology between various pairs of bacteriophages within the lambdoid family and between the tna and tar genes in the Escherichia coli L coli chromosome. Various phage DNA's were annealed in the presence of formamide and the resulting heteroduplex molecules were examined by electron microscopy (Davis and Davidson, 1968; Westmoreland et al, 1969; Davis et al, 1970). Regions of homology in such heteroduplexes appeared as double-stranded segments; regions of nonhomology gave two separate single-stranded segments. Regions of homology and nonhomology were mapped in heteroduplexes made between the DNA's of the various lambdoid coliphages and between A-transducing phages carrying the tr and tna genes of E. coli. This technique was also used to determine the sizes of various DNA molecules and DNA segments. The sizes of the following DNA molecules and DNA fragments were determined: the double-stranded DNA of plasmid pSC101 (19.0 + 0.2%A (s.d.; n = 25)); the single-stranded DNA of bacteriophage M13 (12.7%a); the DNA of bacteriophage A (49,150 ± 730 base pairs (s.d.; n = 25)); the DNA of bacteriophage PA2 (99.6 + 2.0%A (s.d.; n - 19)); the DNA of bacteriophage 424 (96.4 +•1.3%A (s.d.; n = 20)); the HindIII restriction fragment carrying the tna gene of E. coli (12.0 + 0.5%A (s.d.; n = 15)); the HindIII restriction fragment carrying the trpA, trpB and trpC genes of E. coli (11.1 ± 0.4%A (s.d.; n = 38)); the immunity region of a immX) (8.0 + 0.7%A (s.d.; n = 15)); the immunity region of bacteriophage 21 imm21) (3.6 ± 0.4%A (s.d.; n = 15)) and; the nin region of A (5.4 ± O.4%A (s.d.; n = 15)). These values are expressed as the mean ± standard deviation. The number of base pairs comprising A DNA was deduced from the two spreadings A with pSC101 and pSC101 with $X174 (double-stranded); the size of $X174 was determined to be 5386 bases (Sanger et al, 1978). Other DNA segments within the heteroduplexes were also measured. The following heteroduplexes between pairs of lambdoid phages were analyzed x/424, A/PA2, 434/424, 434/21, 434/PA2, 424/PA2 and 21/PA2. The total amount of homology between each pair was 48.3%, 56.7%, 45.4%, 42.7%, 27.8%, 56.6%, 53.3% and 37.4%, respectively. My results indicate that it is likely that the processes envisaged in the modular hypothesis and in the ancestral chromosome hypothesis were important in the evolution of the lambdoid phages. Lambda-transducing phages carrying the trpABC and tna bacterial fragments,generated by restriction of E. coli and A DNA's (with the endonuclease HindIII), followed by ligation and selection for complementation by these genes, were allowed to form heteroduplexes using various denaturing and spreading conditions. The following heteroduplexes were analyzed: NH540/Atna immX c1857 nin+; AtrpABC1/AtrpABCr; A/AtrpABC1; A/AtrpABCr; AtrpABC1/Atna imml cI857 nin-F and; AtrpABC/Atna ion 1857 nin+. The DNA fragments containing the trpABC and tna genes were found to share no more than 58% homology by using the least denaturing conditions possible.