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Analysis of essentiality of glmM gene coding for phosphoglucosamine mutase of Streptococcus pneumoniae.
Krupička, Jiří ; Branny, Pavel (advisor) ; Čáp, Michal (referee)
Phosphoglucosamine mutase (GlmM) is an enzyme of bacterial cell wall biosynthesis. The main aim of this thesis was to find out, whether gene glmM is essential for viability of Streptococcus pneumoniae. Therefore, we prepared merodiploid strain containing two copies of glmM; the genomic gene and ectopic copy under control of zinc inducible promoter. Subsequently, depletion strain was prepared by deletion of genomic copy of glmM. This strain was further used for analysis of viability and phenotype features in the medium containing various concentrations of zinc ions, an inducer of ectopic glmM expression. We found out, that the viability of this strain was strictly dependent on the concentration of inducer and further, that depletion of GlmM resulted in remarkable morphological defects. The rescue of mutant strain was observed after addition of inducer up to the level of the control sample. These results have provided the evidence of glmM essentiality for S. pneumoniae viability. Furthermore, we analyzed, whether phosphorylation of key amino acid residues, S99 and S101, is essential for GlmM functionality. Four different strains were prepared by means of site-directed mutagenesis expressing glmM with substitutions of key serine residues for alanine or glutamic acid. Since deletion of chromosomal locus in...
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Rous sarcoma virus replication blocks in mammalian cells
Koslová, Anna ; Hejnar, Jiří (advisor) ; Ruml, Tomáš (referee) ; Weber, Jan (referee)
One of the important tasks of virology and immunology is to explore the species- and cell-barriers preventing virus horizontal transmission and reveal the ways how viruses overcome these barriers and "adapt" to different species. This work is based on a well- established retroviral model - avian Rous sarcoma virus (RSV) and studies virus replication blocks in mammalian cells at both pre- and post-integration level. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor mediates virus entry into cells. Although mammalian orthologues of specific chicken receptors do not support RSV entry, it was observed that some RSV strains are able to enter mammalian cells. Several RSV-transformed rodent cells lines were described and analysis of provirus H20- RSV in one these cells lines (hamster H-20 tumor cell line) showed multiple mutations including two crucial amino acid substitutions in different regions of Env. Substitutions D32G and L378S confer virus transmission to hamster, human and also chicken cells lacking the appropriate receptor. Altered conformation of H20-RSV Env is similar to a receptor-primed (activated) state of Env. This observation indicates that virus can circumvent the need of original cell receptor because of spontaneous Env activation caused by single...
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Structural ambiguity based on the grammatical indeterminacy of the ing-nonfinite form in subject position
Vojtěch, Albert ; Malá, Markéta (advisor) ; Šaldová, Pavlína (referee)
This thesis deals with the structural ambiguity of constructions similar to Flying planes can be dangerous, where the ambiguity can arise due to the grammatical indeterminacy of ing- nonfinite form that can be interpreted both as gerund and participle. The ambiguity is analysed on the basis of the transitivity of ing-nonfinite form and subsequently on its semantic compatibility with the noun in the role of subject and object, in the ideal case the ambiguity and compatibility is tested further in the sentence context. The first part, the theoretical background presents us with what the secondary sources say to the phenomenon of ambiguity, distinguishes the types of ambiguity and also covers the grammatical description of non-finite verb forms. The Second part, material and method, describes the method of sampling, which brought us 2 samples extracted from British National Corpus. First sample comprises of 25 instances of "ing-nonfinite form + noun + verb constructions" (sg/pl, type of verb and verb tense are not restricted) and the second sample comprises of 100 instances of similar constructions, however the verb following the noun has been restricted on formal grounds for verb tense, verb type and number in order not to represent a hindrance for the ambiguity. The third, analytical part comprises...
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Analysis of essentiality of glmM gene coding for phosphoglucosamine mutase of Streptococcus pneumoniae.
Krupička, Jiří ; Branny, Pavel (advisor) ; Čáp, Michal (referee)
Phosphoglucosamine mutase (GlmM) is an enzyme of bacterial cell wall biosynthesis. The main aim of this thesis was to find out, whether gene glmM is essential for viability of Streptococcus pneumoniae. Therefore, we prepared merodiploid strain containing two copies of glmM; the genomic gene and ectopic copy under control of zinc inducible promoter. Subsequently, depletion strain was prepared by deletion of genomic copy of glmM. This strain was further used for analysis of viability and phenotype features in the medium containing various concentrations of zinc ions, an inducer of ectopic glmM expression. We found out, that the viability of this strain was strictly dependent on the concentration of inducer and further, that depletion of GlmM resulted in remarkable morphological defects. The rescue of mutant strain was observed after addition of inducer up to the level of the control sample. These results have provided the evidence of glmM essentiality for S. pneumoniae viability. Furthermore, we analyzed, whether phosphorylation of key amino acid residues, S99 and S101, is essential for GlmM functionality. Four different strains were prepared by means of site-directed mutagenesis expressing glmM with substitutions of key serine residues for alanine or glutamic acid. Since deletion of chromosomal locus in...
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