No description available.

No description available.

No description available.

The sog locus of ColIb-P9 is known to encode a DNA primase that generates RNA primers for DNA synthesis on a variety of DNA templates and it promotes bacterial DNA replication in primase-defective (dnaG3) mutants of Escherichia coli K-12. The thesis reports that the physiological role of the enzyme is in conjugative metabolism of plasmid DNA. Derivatives of ColIb-P9drd-1 carrying defined sog mutations were constructed by vivo recombination. The mutant plasmids were maintained stably, showing that the primase is inessential for vegetative DNA replication, but they were deficient in transconjugant formation during bacterial mating. Amounts of conjugative DNA synthesis in matings involving mutants and complementing plasmids imply that the primase is required for efficient DNA synthesis on the plasmid strand transferred to the recipient cell, and that the enzyme may act in the donor cell to promote synthesis of a replacement strand. Recipient dnaG3 bacteria, treated with rifampicin to inhibit transcription, recovered some ability to synthesise chromosomal DNA during mating with donors of a Sog+ conjugative plasmid. Recovery was dependent on plasmid primase and an active DNA transfer system but it did not require transmission of a functional sog gene to the recipient cell. It is argued that the recovery reflects conjugative transfer of plasmid primase, that the transferred enzyme normally acts to initiate DNA synthesis on the plasmid strand transmitted from the donor cell, and that sog primase is selectively transferred during conjugation, possibly in association with plasmid DNA. Isolation of recombinant plasmids carrying the origin of transfer or an entry exclusion gene(s) of ColIb-P9drd-l is described.

Hypervariable minisatellite loci provide highly informative genetic markers in mammalian genomes. Hybridisation probes based on a G-rich core sequence simultaneously detect many minisatellite loci in human DNA, generating individual specific and highly variable DNA fingerprints with a wide range of applications. Human minisatellite probes cross-hybridise to mouse DNA, generating DNA fingerprints as complex and variable as those of man. Inbred strains of mice have strain-specific DNA fingerprints which can be analysed using recombinant inbred strains. Analysis of the BXD recombinant inbred series using human minisatellite probes 33.6 and 33.15 revealed that the cross- hybridising loci show variation in germline stability; one locus in particular, Ms6-hm, detected by probe 33.6, exhibited multiallelism across the BXD strains, and heterozygosity among contemporary C57BL/6J inbred mice. Ms6-hm alleles were cloned from C57BL/6J DNA by cross-hybridisation to probe 33.6; on propagation in E.coli the majority of minisatellite repeat units were lost. DNA sequence analysis revealed that Ms6-hm consists of a homogeneous array of the repeat unit GGGCA which has evolved by amplification from within a member of the MT (mouse transcript) family of interspersed repetitive elements. Ms6-hm is flanked by two additional, diverged, MT elements, and there is further evidence that MT elements may be associated with other unstable regions of the mouse genome. Multiallelism and heterozygosity at Ms6-hm (which maps near brown on chromosome 4) result from a high germline mutation rate to new length alleles (2.5% per gamete). Mice mosaic for cells carrying common non-parental Ms6-hm alleles in somatic tissue, and in some cases also in the germline, provide evidence for additional, early developmental, mutation events at this locus. Such somatic mutant Ms6-hm alleles provide innocuous and informative markers with which to analyse the lineage relationships of cells in early mouse development. In four mosaic mice the fraction of cells containing the non-parental allele has been shown to be indistinguishable in different adult tissues, suggesting that the mutation events preceded the allocation of the somatic lineages, and that the same pool of primitive ectoderm cells contributes equally to all somatic tissues. Under low-stringency hybridisation conditions the minisatellite repeat array of Ms6-hm cross-hybridises to other unstable minisatellite loci in the mouse genome to generate a novel and highly individual specific DNA fingerprint; at least one cross-hybridising locus is also somatically unstable during early mouse development.

Mutations at two loci, sfiA and sfiB, suppress the filamentation seen on irradiation of lon mutants or expression of tsl (lexA (Ts)) or tif (recA441) mutations in Escherichia coli. The sfiA and sfiB genes have been assumed to be involved in the inhibition of cell division associated with the SOS response. The product of the sfiA gene has been shown to be a division inhibitor and the sfiB gene has been postulated to be the target for the action of such an inhibitor. The sfiB gene was mapped to the ftsQ-secA region of the E. coli chromosome using P1 transduction and specialised transducing phages. The sfiB+ and sfiB114 genes were cloned onto recombinant plasmids and sfiB114 found to be at least partially dominant in ts1 and lon strains. Using Tn1000 mutagenesis, it was found that the sfiB gene is allelic to the essential septation gene ftsZ and that an element within the preceeding and contiguous ftsA gene is required for full ftsZ (sfiB) expression. Maxi-cells containing plasmids encoding sfiA and either sfiB+ or sfiB114 were used to demonstrate an interaction between SfiA and FtsZ. The presence of an ftsZ(sfiB+) carrying plasmid in maxi-cells increased the half-life of the unstable SfiA protein to 10-14 min (compared to approximately 3 min in the presence of a sfiB114 encoding plasmid or where a sfiA plasmid was present in maxi-cells alone). Finally, maxi-cells containing sfiA and sfiB carrying plasmids were separated into subcellular fractions and it was found that both SfiA and SfiB(FtsZ) proteins bind to the E. coli inner membrane.

Human haemoglobins are coded for by two unlinked clusters of alpha and beta-globin genes. The human beta-globin cluster appears to have evolved by a series of tandem gene duplications and contains considerable stretches of intergenic DNA possibly involved in the developmental control of globin gene expression. In order to understand the evolution of this cluster, the arrangement of primate beta-related globin genes has been determined by restriction endonuclease mapping of genomic DNA from species ranging from prosimians to the great apes and these compared to the published genomic map of the human beta-globin gene cluster. The arrangement of the entire epsilonupsilonupsilonbeta beta-globin gene cluster in the gorilla, chimpanzee and yellow baboon is indistinguishable from that of man. Restriction site differences between these species are consistent with a surprisingly low overall rate of intergenic DNA sequences divergence of approximately 1x10-9 nucleotide substitutions per nucleotide site per year as compared with the currently accepted silent site rate of 5x10-9 nucleotide substitutions per nucleotide site per year. The owl monkey, a New world species, has a single upsilon-globin gene suggesting that the upsilon-globin gene duplication in man is ancient and occurred 20 to 40 million years ago---a surprising contrast to the very high degree of sequence homology existing between these two genes. The owl monkey appears to possess duplicated beta-globin genes suggesting that the deltabeta gene duplication is between 40 and 70 million years old. The beta-globin gene cluster in the brown lemur, a prosimian, is remarkably short (about 17,000 base-pairs) and contains single epsilon-, upsilon- and beta-globin genes. The upsilon- and beta-globin genes in this species are separated by a mosaic gene ([psi]beta') containing the 3' end of a beta-globin gene preceded by sequences related to the 5' end of the epsilon-globin gene as determined by hybridisation studies. The brown lemur beta-globin gene cluster has been cloned into a bacteriophage lambda vector as a set of overlapping DNA fragments covering the entire region and the 'psibeta' gene sub-cloned into a plasmid vector. Initial sequencing the 3' end of this gene has further indicated its beta-like nature.

The approach of gene-fusion was used to analyse genetic control signals. Vectors carrying the trp/lac W205 substitution were constructed to facilitate the fusion vitro of promoters, terminators and other control signals to the lacZ gene of E. coli. Two systems were developed using either a lambda or pBR322 replicon. The plasmid system was developed further to provide probes for promoter and terminator sequences. Restriction analysis of these vectors added to the physical data on the trp/lac W205 fusion. The lambda system was used to analyse transcriptional termination in the b2 region of the phage. This central region was shown to contain terminators insensitive to the anti-terminator proteins specified by the lambda N and Q genes. Gene-fusions were used to examine the control of late gene expression of phage lambda. This expression was dependent on the activity of the gene product. This protein was unusual, being almost completely cis-specific. Transcription of the late genes is initiated in the "6S region" adjacent to the lambda Q gene. This region was cloned into a trp/lac plasmid and its effect was monitored by expression from the lacZ gene fused to this segment. Expression of lacZ from this promoter was limited by a strong terminator 194 base-pairs downstream. However, a weak positive effect was observed in the presence of the lambda Q gene product, mapping the site of Q action, Qat, in or near the 6S region.

An inherent problem in the study of the genetics of the interesting and potentially commercially useful properties of the pseudomonads is that of gene expression, since many of the genes encoding these properties are not well expressed in an E.coli background. The evidence available at the present time indicates that some Pseudomonas genes may possess different promoter sequences not recognised by E.coli RNA polymerase. An in vitro coupled transcription/translation system based on P.putida has been developed. A comparison of E.coli and broad host range plasmid DNA in this and the equivalent E. coli system showed that although cloned E. coli and vector polypeptides were synthesised in both systems, there was a difference in the polypeptide products directed by broad host range plasmid DNA in the two systems. In particular RSF1010 directed the synthesis of a 73kD polypeptide uniquely in the P.putida system. This was shown to be a polypeptide involved in mobilisation of the plasmid. A broad host rsmge proraoter-probe vector based on RSFlOlO was constructed and used for the shotgun cloning of P.putida promoters. A small subset of fragments which were active as promoters in P.putida but exhibited much lower activity in E.coli were isolated, sequenced and analysed with respect to concensus E. coli and nitrogen-regulated promoter sequences. These isolated DNA fragments may represent promoters which have sequences specifically recognised by Pseudomonas RNA polymerase. An analysis of published Pseudomonas chromosomally-encoded promoters revealed putative Pseudomonas-specific concensus regions.

The collagens are a family of structural proteins which function as an extracellular framework in eukaryotic organisms. They are characterised by a unique protein conformation which consists of three polypeptide chains in a triple helix. In vertebrates, at least thirteen collagen types encoded by at least twenty non-allelic genes have been identified. The collagen genes constitute a multi-gene family with a probable common evolutionary origin. Though generally dispersed, some of the genes are known to be clustered on certain chromosomes. Alterations in collagen genes can result in a heterogeneous group of heritable disorders of the connective tissues. There is accumulating evidence that similar phenotypes are due to similar mutations or location of mutations. One inherited disease of the connective tissues is the Marfan syndrome (MS). The genetic basis of this disease is still unknown. The four genes of the fibrillar collagens types I, II and III were chosen as candidate genes for a study of linkage in a large MS family. Three of these genes were excluded as being the mutant gene in this family. The heritable brittle bone disease, osteogenesis imperfecta (OI) is usually caused by mutations in either of the two genes for type I collagen. The technique of RNase A cleavage mapping was used to search for mutations in the a1 (I) gene in the lethal type of OI. The mutation in one individual was detected and mapped to exon 43 of the a1 (I) gene (COL1A1). The mutant gene was amplified, cloned and sequenced revealing a 9bp deletion from a repetitive region of one allele. It is surprising that such a mutation could produce OI in its most severe form. Studies to verify that the deletion is the disease-cause remain to be undertaken.