Expression of streptomycete cholesterol oxidase inEscherichia coli

Summary A streptomycete gene coding for extracellular cholesterol oxidase (choA) was subcloned and expressed inEscherichia coli. The pUCO series recombinants were obtained by inserting thechoA gene into the uniqueKpnI site of pUC19 vector. Expression was observed with pUCO192A and pUCO193 constructs in which the cloned gene(s) were aligned with the upstreamlacZ promoter. Isopropyl -d-thioglucopyranoside (IPTG) enhanced this expression up to 2.5-fold. Specific Cho activity in the cell extracts of the stable pUCO193 transformant were 0.004 U and 0.007 U per mg protein without and with IPTG induction, respectively. Cho activity was detected in the spent medium of this culture, suggesting possible secretion of the enzyme.     

Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters

We have tested the use of firefly luciferase for monitoring regulated symbiotic nitrogen fixation gene expression. Broad-host-range plasmids carrying translational fusions of Rhizobium meliloti nifH, fixA and nifA promoters were constructed. Despite low levels of promoter activity the absence of Escherichia coli endogenous luminescence and the high sensitivity of the bioluminescent assay for firefly luciferase allowed rapid screening for functional luciferase expression. Plasmids containing symbiotic promoter-luc fusions were established in R. meliloti. Luciferase activity was detected and measured in both vegetative and symbiotic cells giving comparable results with those obtained by beta-galactosidase assays. In addition, the luciferase assay was quicker, more sensitive and could be carried out with unrestricted cells. Furthermore, bioluminescence was high enough in alfalfa nodules containing nifH—luc fusion to be observed by a dark-adapted eye and photographed.     

Formation of functionally active chloroplasts is determined at a limited stage of leaf development in virescent mutants of rice

The ability to form functionally active chloroplasts is determined at a certain early stage of leaf development in three non-allelic temperature-sensitive virescent mutants of rice. Temperature-shift analysis, together with anatomical observations, indicates that the intrinsic developmental signals of the virescent genes are expressed at the stage immediately following the formation of basic leaf structure, but just before the onset of leaf elongation. These signals control the expression of chloroplast-encoded genes but do not affect the subsequent morphological development of the leaf or the photo-regulation of the expression of nuclear genes encoding chloroplast proteins.     

Chaos game representation of coding regions of human globin genes and alcohol dehydrogenase genes of phylogenetically divergent species

Summary Chaos game representation (CGR) is a novel holistic approach that provides a visual image of a DNA sequence quite different from the traditional linear arrangement of nucleotides. Although it is known that CGR patterns depict base composition and sequentiality, the biological significance of the specific features of each pattern is not understood. To systematically examine these features, we have examined the coding sequences of 7 human globin genes and 29 relatively conserved alcohol dehydrogenase (Adh) genes from phylogenetically divergent species. The CGRs of human globin cDNAs were similar to one another and to the entire human globin gene complex. Interestingly, human globin CGRs were also strikingly similar to human Adh CGRs. Adh CGRs were similar for genes of the same or closely related species but were different for relatively conserved Adh genes from distantly related species. Dinucleotide frequencies may account for the self-similar pattern that is characteristic of vertebrate CGRs and the genome-specific features of CGR patterns. Mutational frequencies of dinucleotides may vary among genome types. The special features of CG dinucleotides of vertebrates represent such an example. The CGR patterns examined thus far suggest that the evolution of a gene and its coding sequence should not be examined in isolation. Consideration should be given to genome-specific differential mutation rates for different dinucleotides or specific oligonucleotides. Offprint requests to: S. M. Singh     

Morphometric Analysis of Normal, Mutant, and Transgenic CNS: Correlation of Myelin Basic Protein Expression to Myelinogenesis

The neurological mutant mice shiverer (shi) and myelin deficient (shimld) lack a functional gene for the myelin basic proteins (MBP), have virtually no myelin in their CNS, shiver, seize, and die early. Mutant mice homozygous for an MBP transgene have MBP mRNA and MBP in net amounts approximately 25% of normal, have compact myelin, do not shiver or seize, and live normal life spans. We bred mice with various combinations of the normal, transgenic, shi, and shimld genes to produce mice that expressed MBP mRNA at levels of 0, 5, 12.5, 17.5, 50, 62.5, and 100% of normal. The CNS of these mice were analyzed for MBP content, tissue localization of MBP, degree of myelination, axon size, and myelin thickness. MBP protein content correlated with predicted MBP gene expression. Immunocytochemical staining localized MBP to white matter in normal and transgenic shi mice with an intensity of staining comparable to the degree of MBP gene expression. An increase in the percentage of myelinated axons and the thickness of myelin correlated with increased gene expression up to 50% of normal. The percentage of myelinated axons and myelin thickness remained constant at expression levels greater than 50%. The presence of axons loosely wrapped with oligodendrocytic membrane in mice expressing lower amounts of MBP mRNA and protein suggested that the oligodendroglia produced sufficient MBP to elicit axon wrapping but not enough to form compact myelin. Mean axon circumference of myelinated axons was greater than axon circumference of unmyelinated axons at each level of gene expression, further evidence that oligodendroglial cells preferentially myelinate axons of larger caliber.(ABSTRACT TRUNCATED AT 250 WORDS)