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When a cell divides into two, it is imperative that each resultant daughter receives a full complement of chromosomes; DNA is ultimately responsible for all cellular processes. Cohesion between sister chromatids from the moment of their generation in S phase is central to ensuring the fidelity of chromosome segregation. Smc1 and Smc3 proteins interact with each other via their hinges and with a bridging kleisin subunit via their heads to form the cohesin ring. It is cohesin, through entrapment of sister chromatid within its ring, that confers sister chromatid cohesion. The process of cohesin’s loading onto DNA is poorly understood. While it is thought to depend on ATP hydrolysis, opening of the ring at one of its three interfaces, and the as yet undefined action of the kollerin complex, comprising Scc2 and Scc4 proteins, the sequence of events as they occur are yet to be defined. A recent screen for suppressors of a thermosensitive scc4 allele in budding yeast revealed a mutation within Smc1’s hinge that could bypass the kollerin subunit. Here, the Smc1 suppressor mutation is investigated. Through targeted mutagenesis, the Smc1D588Y mutant identified in the screen and two additional point mutants, Smc1D588F and Smc1D588W, are herein proven able to bypass Scc4 function completely. Thus we provide the strongest evidence to date to suggest that cohesin’s hinge is a critical factor in its loading. Biochemical evidence shows that isolated Smc1 hinge mutants are defective in their binding to Smc3 hinges. This, together with the genetic link made between the hinge and loading complex, suggests that hinge opening might be a requisite for loading. Through mutagenesis of Scc2 and Scc4 we show that the N-terminus of each protein is responsible for their dimerisation. Furthermore, the N- terminus of Scc2 confers no function other than in its binding to Scc4. Finally, we show that Scc4 is required for the enrichment of both Scc2 and cohesin at centromeres, but not at arm loci. Our results are therefore indicative of there being two different pathways of cohesin loading.

Numerous current environmental problems result from changes to the trace gas composition of the Earth's atmosphere. Issues such as smog formation, acid rain and stratospheric ozone depletion all result from the chemical transformation of pollutants in the atmosphere and understanding the nature and rapidity of these chemical changes is consequently an important goal of atmospheric science. Laboratory studies of the kinetics and product channels of important atmospheric reactive species - atoms and free radicals - aim to provide data for inclusion into numerical models to simulate changing atmospheric compositioa Typically, the limitation of such laboratory studies is the sensitivity required for monitoring very low concentrations of very short-lived gas phase species. The work described in this thesis addresses mislimitation in new instrument development and in application of an existing instrument to studies of reactions of the atmospherically important CIO free radical. This thesis therefore describes two principal areas of research. In the first part, the design, construction, and testing of an experimental technique for the study of gas phase chemical reactions in a flow system are detailed. This apparatus consists of a conventional discharge flow tube in which reactions take place, coupled to a novel detection system This detection, based upon chemical ionisation mass spectrometry (CIMS), uses a novel chemical ionisation scheme using proton transfer from H3+ ions. A high-pressure electrical discharge system using H2 has been developed and used as the source of H3+ An electrostatic ion guide has also been designed and tested to transport ions efficiently from their region of interaction with the gas under study, to the detector. Experiments to characterise the detection efficiency and instrumental sensitivity have been carried out, along with studies of a variety of chemical systems.